Biologic compounds

ABSTRACT

The present invention relates to amino acid sequences that are directed against TRAIL cell surface receptor 2 (herein also “DR5”), as well as to compounds or constructs thereof, and in particular proteins and polypeptides and nucleotides that encode them (referred to herein in their entirety as “NB agents”) and fragments thereof, and pharmaceutically effective variants thereof, and their use in the diagnosis and treatment of DR5 associated diseases and disorders.

BACKGROUND

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand(TRAIL) induces apoptosis in a variety of tumorigenic and transformedcell lines with little or no effect on normal cells. At least fivereceptors for TRAIL have been identified, of which two, namely DR4(Death Receptor 4, TRAIL-R1) and DR5 (Death Receptor 5, TRAIL-R2; KILLERor TRICK 2), are capable of transducing an apoptosis signal, whereas theother three (TRAIL-R3, TRAIL-R4 and soluble OPG) serve as decoyreceptors to block TRAIL-mediated apoptosis. See e.g., Ozoren andEl-Deiry, Sem. Cancer Biol 13: 135 (2003); Yagita et al., Cancer Sci.,95(10): 777 (2004).

TRAIL or Apo2L is a 281 amino-acid cytotoxic ligand found integratedinto the cytoplasmic membrane with the C-terminus exposed at theextracellular surface (Type II ligand) of cells. Small quantities ofsoluble TRAIL ligand can also be detected. TRAIL forms a homo-trimericmolecule that binds its respective receptors, initiating a cascade ofsignaling events.

The binding of TRAIL ligand to the receptors DR4 or DR5 initiates theextrinsic cell death pathway, resulting in the formation ofdeath-inducing signaling complexes (DISC), which contain the adaptorFADD (Fas-activating DD) and pro-caspase 8 or pro-caspase 10. Theinteractions at the DISC and the activation of the downstream cascadeare similar to FAS, resulting in activation of the NFκB and JunN-terminal kinase pathways (JNK). See, e.g., Mongkolsapaya et al., Nat.Struct. Biol., 6(11): 1048 (1999); Cha et al., J. Biol. Chem., 275(40):31171 (2000). TRAIL binding to DR4 or DR5 also results in a BID cleavage(by caspase 8 or 10), activation of mitochondria and hence activation ofthe intrinsic apoptosis pathway.

The ability of TRAIL ligand to preferentially induce apoptosis of tumorcells, with little or no effect on normal cells, makes it a potentiallygood candidate for cancer therapy. However, soluble TRAIL has been shownto induce apoptosis of normal human hepatocytes in vitro, highlightingpotentially toxicity concerns. See, e.g., Jo et al., Nat Med. 6(5): 564(2000). A more advantageous target for cancer therapy is DR5. BecauseDR5 requires multiple receptors to induce apoptosis of normalhepatocytes, the development of agonists against the specific DR5responsible for induction of apoptosis is predicted to avoid thistoxicity against normal cells, while retaining the ability to kill tumorcells.

Peptide and antibody agonists of DR5 have been used to induce apoptosisin cells expressing DR5. See, e.g., Li et al., J. Mol. Biol., 361,522-536 (2006); Kajiwara et al., Biochim. Biophys. Acta 1699: 131-137(2004); Yang et al., Cancer Cell, 5, 501-512 (2004). Agonists of DR5have the potential to be used in cancer therapy against a wide range ofcancers. For example, Lexatumumab, a monoclonal antibody against DR5,induces expression of DR5 and promotes apoptosis in a mouse model ofrenal cell carcinoma. Zhang et al., Cancer Lett. 251(1): 146-57 (2007).Other agonistic monoclonal antibodies against DR5, or single-chain Fvfragment against DR5, and the tumoricidal activity thereof are similarlydescribed, e.g., in Takeda et al., Journal Exp. Medicine, 199, 437-448(2004); Guo et al., J. Biol. Chem., 280, 41940-41952 (2005); Motoki etal., Clin. Cancer Res 11(8): 3126-35 (2005); and Ichikawa et al., NatureMedicine, 7, 954-960 (2001), Chuntharapai et al., J. Immunol. 166(8):4891-8 (2001), Shi et al., Cancer Res., 66(24): 11946-11953 (2006).

Various DR5 specific antibodies are being developed for use in theclinic but none are yet approved. It is believed that for DR5 to beactivated, multiple cross-linking antibodies are required, and issuesremain for sufficient delivery of these antibodies at the site of actionto achieve reproducible therapeutic effects. Therefore, a need existsfor improved DR5 specific agonists for treatment of associated diseases.

SUMMARY OF THE INVENTION

The present invention relates to one or more “NB agents” comprisingamino acid sequences that are directed against Death Receptor 5 (hereinreferred to as “DR5”) as provided herein, as well as to specificcompounds or constructs, and in particular proteins and polypeptides,that comprise or essentially consist of one or more such amino acidsequences (also referred to as “compounds”, and “polypeptides”,respectively) and fragments thereof, plus the nucleic acids that encodesuch constructs. Monomeric and multimeric variants of the inventive DR5specific constructs are provided herein. Humanized and humaneeredvariants of the NB agents are provided. In one embodiment,pharmaceutically relevant NB agents of the invention act as agonists ofthe TRAIL receptors DR5.

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides; methods for preparing such amino acidsequences and polypeptides; host cells expressing or capable ofexpressing such amino acid sequences or polypeptides; compositions, andin particular to pharmaceutical compositions, that comprise such aminoacid sequences, polypeptides, nucleic acids and/or host cells; and usesof such amino acid sequences or polypeptides, nucleic acids, host cellsand/or compositions, in particular for prophylactic, therapeutic ordiagnostic purposes, such as the purposes mentioned herein.

Agonists of the TRAIL receptors DR5 described herein as NB agents havethe potential to be used in therapy against a wide range of diseasesassociated with DR5. These inventive NB agents therefore have utility inthe treatment of proliferative diseases including, e.g., cancers such assolid tumors, primary and metastatic cancers such as renal cellcarcinoma, and cancers of the lung (e.g., small cell lung cancer “SCLC”and non-small cell lung cancer “NSCLC”), pancreas, hematopoieticmalignancy, glioma, astrocytoma, mesothelioma, colorectal cancers,prostate cancer, osteosarcoma, melanoma, lymphoma (including but notlimited to Burkitt's Lymphoma), breast cancer, endometrial cancer, livercancer, gastric cancer, skin cancer, ovarian cancer and squamous cellcancers of any origin (e.g., lung, head and neck, breast, thyroid,cervix, skin, esophageal, etc.), as well as liquid cancers, e.g., suchas leukemias (see, e.g., Uno et al., Nature Medicine, 12(6):693-698(2006)) including especially a T-cell leukemia such as acuteT-cell leukemia (T-ALL), acute B-cell leukemia (B-ALL), chronicmyelogenous leukemia (CML), acute myelogenous leukemia (AML), plasmacell myeloma and multiple myeloma (MM). These inventive NB agents alsohave utility in the treatment of non-cancer indications includeinflammatory and autoimmune disease, such as systemic lupuserythematosus, Hashimoto's disease, rheumatoid arthritis,graft-versus-host disease, Sjogren's syndrome, pernicious anemia,Addison disease, scleroderma, Goodpasture's syndrome, Crohn's disease,autoimmune hemolytic anemia; sterility, myasthenia gravis, multiplesclerosis, Basedow's disease; thrombotic throbocytopenia, thrombopeniapurpurea, insulin-dependent diabetes mellitus, allergy; asthma; atopicdisease; arteriosclerosis; myocarditis; cardiomyopathy; globerulanephritis; and hypoplastic anemia.

The amino acid sequences and polypeptides described herein may bedirected against, or be specific to, any DR5. According to onenon-limiting aspect, amino acid sequences and polypeptides describedherein are directed against DR5. In one embodiment, the NB agentsspecifically bind to a DR5 extracellular domain.

The polypeptides and compositions of the present invention can generallybe used to modulate DR5. In one embodiment, an NB agent will trigger,activate and/or increase or enhance the signaling that is mediated byDR5. In one embodiment, agonism of DR5 by an NB agent will modulate, andin particular trigger or increase the biological mechanisms, responsesand effects associated with DR5, their signaling and/or the pathways inwhich DR5 is involved.

In one embodiment, the compounds, polypeptides and compositionsdescribed herein induce, trigger, increase or enhance apoptosis incertain cells or tissues. In one embodiment, the compounds, polypeptidesand compositions described herein are capable of binding to DR5 on acell surface, and in particular of binding to a DR5 in such a way thatthe signaling mediated by the DR5 is induced, triggered, increased orenhanced. In one embodiment, the polypeptides and compositions describedherein may be such that they are capable of binding to a DR5 in such away that apoptosis is triggered or induced in the cell on which the DR5is present.

In one embodiment, the NB compounds, (monovalent or multivalent)polypeptides and compositions described herein can bind to the bindingsite of TRAIL on the DR5. In one embodiment, the NB agents compete withTRAIL for binding to the DR5. In one embodiment, the binding of suchcompounds and polypeptides to DR5 induces, triggers, increases orenhances the signaling mediated by the DR5, and in particular triggersor induces apoptosis in the cell on which the DR5 is present.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of an ELISA experiment showing thebinding of multivalent anti-DR5 NB agents against TNF Receptor familymembers.

FIG. 2 is a graphic representation of a tumor tissue PD (Caspase3/7activation) of tetravalent anti-DR5 NB agents.

FIG. 3 is a graphic representation of the in vivo efficacy oftetravalent and pentavalent anti-DR5 NB agents in a Colo205 tumorsmodel.

FIGS. 4A and 4B are graphic representations of 11H6 tetramer exhibitinganti-tumor activity in a MiaPaCa pancreatic tumor model grown in NSGhosts (i.e., macrophage/NK deficient hosts) either without (FIG. 4A) orwith (FIG. 4B) CSF-1R inhibitor.

FIG. 5 shows 11H6 tetramer ability to regress patient-derived pancreatictumor TPAN1-IFA that is insensitive to LCR211, a murine antibodyspecific to DR5.

FIGS. 6A, 6B and 6C are graphic representations showing that anincreased number of subunits in a NB construct corresponds with animproved efficacy and potency for (FIG. 6A) 11H6 trimers, tetramers andpentamers in Colo205 cells; (FIG. 6B) 4E6 and 11H6 tetramers andpentamers in Colo205 cells; and (FIG. 6C) 4E6 and 11H6 tetramers andpentamers in H226 cells.

DETAILED DESCRIPTION OF THE INVENTION

The NB agents described herein, namely the compounds, polypeptides(monovalent or multivalent) and compositions, bind to Death Receptor 5(“DR5”). In one embodiment, the binding of the disclosed compounds andpolypeptides to the DR5 induces, triggers, increases or enhances thesignaling mediated by the DR5, and in particular triggers or inducesapoptosis in the cell on which the DR5 is present.

As used herein a “NB agent” of the invention is defined as a polypeptidecontaining at least one CDR3 sequence that specifically bind to a DR5polypeptide and whose binding produces an agonistic effect on DR5activity, or a nucleotide that encodes such a polypeptide. In oneembodiment, NB agents of the invention are a polypeptide having thegeneral structure of a V_(H) domain. In one embodiment, NB agents of theinvention are a polypeptide having the general structure of a V_(HH)domain. In one embodiment, NB agents of the invention are a polypeptidehaving the general structure of a V_(L) domain. In one embodiment, NBagents of the invention contain at least one CDR1 and at least one CDR2sequence in addition to the CDR3 sequence. In one embodiment, NB agentsof the invention comprise at least one monovalent polypeptide having theFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 construction as defined below forFormula 1. In one embodiment, NB agents of the invention are monomericcompositions. In one embodiment, NB agents are multimeric compositions.In one embodiment, NB agents of the invention are monovalentcompositions. In one embodiment, NB agents are multivalent compositions.In one embodiment, the framework regions contain residues that aresequence optimized for use in humans. Other aspects of NB agents of theinvention are described below.

Whereas the term “NB agent” is used as a general term that is meant toencompass the full scope of the compositions of the invention, the terms“NB sequence” or alternatively “NB construct” are used herein to referto the specific polypeptide variants disclosed herein as SEQ ID NOS:1-72, 87-88 and 102-103 and the nucleotides that encode them, includingthose specifically disclosed herein as SEQ ID NOS: 96-99 and thosesequences that encode the same polypeptide sequences as SEQ ID NOs:96-99 but differ by the degenerate code.

NB Agents of the Invention

In one embodiment, the NB agent is selected from the group of 11D1,11H6, 10F1, 7A12, and 4E6, or a variant thereof. In one embodiment, theNB agent is a variant whose sequence is optimized for use in humans,e.g., is humanized. In one embodiment humanized NB agents includewithout limitation the group of 4E6hu and 11H6hu. In one embodiment, theNB agent is a monomeric variant of the listed group, or a variantthereof. In one embodiment, the NB agent is a multimeric variant of thelisted group, or a variant thereof. In one embodiment, the multimericvariant of the listed group is monospecific, or a variant thereof. Inone embodiment, the multimeric variant of the listed group ismultispecific, or a variant thereof. In one embodiment, the multipleepitopes recognized by a multispecific NB agent are various epitopes ofthe DR5 target, or a variant thereof. In one embodiment, the multipleepitopes recognized by a multispecific NB agent is at least one epitopeof the DR5 target and at least one epitope on a target other than theDR5 polypeptide, or a variant thereof.

In one embodiment, the NB agent is selected from one or more of SEQ IDNOs: 1-22, 26-40, 87-88, and 102-103, or SEQ ID NOs: 96-99, or a variantthereof. Specific embodiments of NB agents 11D1, 11H6, 10F1, 7A12, and4E6, or variants thereof, are as provided below and in Tables 1-4.

Exemplary NB agents of the invention include the following NBpolypeptide constructs and variants thereof.

4E6 Family

In one embodiment, the NB agent is 4E6, or a variant thereof. In oneembodiment, the 4E6 construct is the monomer of SEQ ID NO: 1, or avariant thereof. In one embodiment, the 4E6 construct is the monomer ofSEQ ID NO: 26, or a variant thereof. In one embodiment, the 4E6construct is a multimer comprising two or more monomers of SEQ ID NO: 1that are operably linked, or a variant thereof. In one embodiment, the4E6 construct is a multimer comprising two or more monomers of SEQ IDNO: 26 that are operably linked, or a variant thereof. In oneembodiment, the 4E6 construct is a multimer comprising two or moremonomers of both SEQ ID NO: 1 and of SEQ ID NO: 26 that are operablylinked, or a variant thereof, wherein the monomers may be in any order.

In one embodiment, the 4E6 multimeric construct is a trimer, a tetrameror a pentamer of SEQ ID NO: 1, or a variant thereof. In one embodiment,the 4E6 multimeric construct is a trimer, a tetramer or a pentamer ofSEQ ID NO: 26, or a variant thereof. In one embodiment, the 4E6multimeric construct is a trimer, a tetramer or a pentamer of acombination of both SEQ ID NO: 1 and of SEQ ID NO: 26, or a variantthereof, wherein the monomers may be in any order. In one embodiment,the 4E6 construct is the multimer as provided in any one or more of SEQID NOs: 6-8 and 27-29, or a variant thereof. Higher order multimers,such as multimers comprised of 6-10 subunits, are also contemplated.

In one embodiment, the 4E6 construct comprises SEQ ID NO: 64, or avariant thereof. In one embodiment, the 4E6 construct comprises SEQ IDNO: 65, or a variant thereof. In one embodiment, the 4E6 constructcomprises a CDR1 sequence of SEQ ID NO: 42, a CDR2 sequence of SEQ IDNO: 52 and a CDR3 sequence of any one or more of SEQ ID NO: 64 and/orSEQ ID NO: 65, or a variant thereof. In one embodiment, the 4E6construct is a humanized or humaneered sequence, or a variant thereof.In one embodiment, the 4E6 construct is as provided in SEQ ID NO: 28, ora variant thereof. In one embodiment, the 4E6 construct is as providedin SEQ ID NO: 29, or a variant thereof. In one embodiment, the 4E6construct comprises SEQ ID NOs: 35, 42, 46, 52, 57, 64 and 70,respectively, or a variant thereof. In one embodiment, the 4E6 constructcomprises SEQ ID NOs: 36, 42, 47, 52, 58, 65 and 71, respectively, or avariant thereof.

In one embodiment, the NB construct is a nucleic acid that encodes the4E6 polypeptide of SEQ ID NO: 1 or variants, multimers or fragmentsthereof. In one embodiment, the NB construct is a nucleic acid thatencodes the 4E6Hu polypeptide of SEQ ID NO: 26 or variants, multimers orfragments thereof. In one embodiment, the NB construct is a nucleic acidthat encodes one or more of the CDR regions of the 4E6Hu polypeptide,namely SEQ ID NO: 42, 52 and 64, or variants thereof. In one embodiment,the NB construct is a nucleic acid that encodes one or more of the CDRregions of the 4E6 polypeptide, namely SEQ ID NO: 42, 52 and 65, orvariants thereof. In one embodiment, the NB construct is a nucleic acidthat encodes the CDR3 region of SEQ ID NO: 64 or variants thereof. Inone embodiment, the NB construct is a nucleic acid that encodes the CDR3region of SEQ ID NO: 65 or variants thereof.

7A12 Family

In one embodiment, the NB agent is 7A12, or a variant thereof. In oneembodiment, the 7A12 construct is the monomer of SEQ ID NO: 2, or avariant thereof. In one embodiment, the 7A12 construct is a multimercomprising two or more monomers of SEQ ID NO: 2 that are operablylinked, or a variant thereof. In one embodiment, the 7A12 multimericconstruct is a trimer, a tetramer or a pentamer of SEQ ID NO: 2, or avariant thereof. In one embodiment, the 7A12 construct is the multimeras provided in any one or more of SEQ ID NOs: 9-11, or a variantthereof. In one embodiment, the 7A12 construct comprises SEQ ID NO: 63.In one embodiment, the 7A12 construct comprises a CDR1 sequence of SEQID NO: 41, a CDR2 sequence of SEQ ID NO: 51 and a CDR3 sequence of SEQID NO: 63, or a variant thereof. In one embodiment, the 7A12 constructis a humanized or humaneered sequence, or a variant thereof. In oneembodiment, the 7A12 construct is as provided in SEQ ID NO: 10, or avariant thereof. In one embodiment, the 7A12 construct is as provided inSEQ ID NO: 11, or a variant thereof. In one embodiment, the 7A12construct comprises SEQ ID NOs: 34, 41, 45, 51, 56, 63 and 69,respectively, or a variant thereof. Higher order multimers, such asmultimers comprised of 6-10 subunits, are also contemplated.

In one embodiment, the NB construct is a nucleic acid that encodes the7A12 polypeptide of SEQ ID NO: 2 or variants, multimers or fragmentsthereof. In one embodiment, the NB construct is a nucleic acid thatencodes one or more of the CDR regions of SEQ ID NO: 41, 51 and 63, orvariants thereof. In one embodiment, the NB construct is a nucleic acidthat encodes the CDR3 region of SEQ ID NO: 63 or variants thereof.

10F1 Family

In one embodiment, the NB agent is 10F1, or a variant thereof. In oneembodiment, the 10F1 construct is the monomer of SEQ ID NO: 3, or avariant thereof. In one embodiment, the 10F1 construct is a multimercomprising two or more monomers of SEQ ID NO: 3 that are operablylinked, or a variant thereof. In one embodiment, the 10F1 multimericconstruct is a trimer, a tetramer or a pentamer of SEQ ID NO: 3, or avariant thereof. In one embodiment, the 10F1 construct is the multimeras provided in any one or more of SEQ ID NOs: 12-16, or a variantthereof. In one embodiment, the 10F1 construct comprises SEQ ID NO: 68,or a variant thereof. In one embodiment, the 10F1 construct comprises aCDR1 sequence of SEQ ID NO: 44, a CDR2 sequence of SEQ ID NO: 55 and aCDR3 sequence of SEQ ID NO: 68, or a variant thereof. In one embodiment,the 10F1 construct is a humanized or humaneered sequence, or a variantthereof. In one embodiment, the 10F1 construct comprises SEQ ID NO: 15,or a variant thereof. In one embodiment, the 10F1 construct comprisesSEQ ID NO: 16, or a variant thereof. In one embodiment, the 10F1construct comprises SEQ ID NOs: 40, 44, 50, 55, 62, 68 and 72,respectively, or a variant thereof. Higher order multimers, such asmultimers comprised of 6-10 subunits, are also contemplated.

In one embodiment, the NB construct is a nucleic acid that encodes the10F1 polypeptide of SEQ ID NO: 3 or variants, multimers or fragmentsthereof. In one embodiment, the NB construct is a nucleic acid thatencodes one or more of the CDR regions of SEQ ID NO: 44, 55 and 68, orvariants thereof. In one embodiment, the NB construct is a nucleic acidthat encodes the CDR3 region of SEQ ID NO: 68 or variants thereof.

11D1 Family

In one embodiment, the NB agent is 11D1, or a variant thereof. In oneembodiment, the 11D1 construct is the monomer of SEQ ID NO: 4, or avariant thereof. In one embodiment, the 11D1 construct is a multimercomprising two or more monomers of SEQ ID NO: 4 that are operablylinked, or a variant thereof. In one embodiment, the 11D1 multimericconstruct is a trimer, a tetramer or a pentamer of SEQ ID NO: 4, or avariant thereof. In one embodiment, the 11D1 construct is the multimeras provided in any one or more of SEQ ID NOs: 17-19, or a variantthereof. In one embodiment, the 11D1 construct comprises SEQ ID NO: 67,or a variant thereof. In one embodiment, the 11D1 construct comprises aCDR1 sequence of SEQ ID NO: 43, a CDR2 sequence of SEQ ID NO: 54 and aCDR3 sequence of SEQ ID NO: 67, or a variant thereof. In one embodiment,the 11D1 construct is a humanized or humaneered sequence, or a variantthereof. In one embodiment, the 11D1 construct comprises SEQ ID NO: 18,or a variant thereof. In one embodiment, the 11D1 construct comprisesSEQ ID NO: 19, or a variant thereof. In one embodiment, the 11D1construct comprises SEQ ID NOs: 39, 43, 49, 54, 61, 67 and 71,respectively, or a variant thereof. Higher order multimers, such asmultimers comprised of 6-10 subunits, are also contemplated.

In one embodiment, the NB construct is a nucleic acid that encodes the11D1 polypeptide of SEQ ID NO: 4 or variants, multimers or fragmentsthereof. In one embodiment, the NB construct is a nucleic acid thatencodes one or more of the CDR regions of SEQ ID NO: 43, 54 and 67, orvariants thereof. In one embodiment, the NB construct is a nucleic acidthat encodes the CDR3 region of SEQ ID NO: 67 or variants thereof.

11H6 Family

In one embodiment, the NB agent is 11H6, or a variant thereof. In oneembodiment, the 11H6 construct is the monomer of SEQ ID NO: 5, or avariant thereof. In one embodiment, the 11H6 construct is the monomer ofSEQ ID NO: 30, or a variant thereof. In one embodiment, the 11H6construct is a multimer comprising two or more monomers of SEQ ID NO: 5that are operably linked, or a variant thereof. In one embodiment, the11H6 construct is a multimer comprising two or more monomers of SEQ IDNO: 30 that are operably linked, or a variant thereof. In oneembodiment, the 11H6 construct is a multimer comprising two or moremonomers of both SEQ ID NO: 5 and of SEQ ID NO: 30 that are operablylinked, or a variant thereof (e.g., SEQ ID NO: 88), wherein the monomersmay be in any order. Higher order multimers, such as multimers comprisedof 6-10 subunits, are also contemplated.

In one embodiment, the 11H6 multimeric construct is a trimer, a tetrameror a pentamer of SEQ ID NO: 5, or a variant thereof. In one embodiment,the 11H6 multimeric construct is a trimer, a tetramer or a pentamer ofSEQ ID NO: 30, or a variant thereof. In one embodiment, the 11H6multimeric construct is a trimer, a tetramer or a pentamer of acombination of both SEQ ID NO: 5 and of SEQ ID NO: 30, or a variantthereof, wherein the monomers may be in any order. In one embodiment,the 11H6 construct is the multimer as provided in any one or more of SEQID NOs: 20-22 and 31-33, or a variant thereof.

In one embodiment, the 11H6 construct comprises SEQ ID NO: 66 or 83, ora variant thereof. In one embodiment, the 11H6 construct comprises aCDR1 sequence of SEQ ID NO: 43, a CDR2 sequence of SEQ ID NO: 53 and aCDR3 sequence of any one or more of SEQ ID NO: 66, or a variant thereof.In one embodiment, the 11H6 construct is a humanized or humaneeredsequence, or a variant thereof. In one embodiment, the 11H6 construct isas provided in SEQ ID NO: 31, or a variant thereof. In one embodiment,the 11H6 construct is as provided in SEQ ID NO: 32, or a variantthereof. In one embodiment, the 11H6 construct is as provided in SEQ IDNO: 33, or a variant thereof. In one embodiment, the 11H6 constructcomprises SEQ ID NOs: 37, 43, 48, 53, 59, 66 and 70, respectively, or avariant thereof. In one embodiment, the 11H6 construct comprises SEQ IDNOs: 38, 43, 48, 53, 60, 66 and 71, respectively, or a variant thereof.

In one embodiment, the NB construct is a nucleic acid that encodes the11H6 polypeptide of SEQ ID NO: 5 or variants, multimers or fragmentsthereof. In one embodiment, the NB construct is a nucleic acid thatencodes the 11H6Hu polypeptide of SEQ ID NO: 30 or variants, multimersor fragments thereof. In one embodiment, the NB construct is a nucleicacid that encodes one or more of the CDR regions of SEQ ID NO: 43, 53and 66, or variants thereof. In one embodiment, the NB construct is anucleic acid that encodes the CDR3 region of SEQ ID NO: 66 or variantsthereof.

Other Contemplated Variants of NB Agents

In one embodiment, a NB agent of the invention is a multimeric constructcomprising at least two different subunits selected from the group of11D1, 11H6, 10F1, 7A12, and 4E6, or a variant thereof. In one suchembodiment, the particular order of the multimeric constructs comprisesalternating subunits. An exemplary construct is provided in SEQ ID NO:84. Other arrangements of the subunits within a multimeric NB constructare contemplated. A skilled person will generally be able to determineand select suitable substitutions, deletions or insertions, or suitablecombinations of thereof, based on the disclosure herein and optionallyafter a limited degree of routine experimentation, which may e.g.,involve introducing a limited number of possible substitutions anddetermining their influence on the properties of the NB constructs thusobtained.

In addition to the disclosed exemplary NB polypeptides constructsprovided herein, the invention encompasses nucleotides that encode anyNB agent encompassed within the present invention disclosure, includingwithout limitation: DNA and RNA sequences or variants thereof, inisolation or in recombinant vectors or the like, and/or as codingregions or fragments including especially fragments encoding the CDR3region of the disclosed NB constructs. In one embodiment, the NB agentis a nucleic acid that encodes the NB construct of any one of thesequences provided in Tables 1-4. In one embodiment, the NB agent is anucleic acid that encodes the 4E6 construct of any one of SEQ ID NOS:1-5, 2, 3, 4, 5, 26, 30 and 87. In one embodiment, the NB agent is anucleic acid that encodes any one of SEQ ID NOS: 41-44, 51-55 and 63-68.In one embodiment, the NB agent is a nucleic acid that encodes any oneof SEQ ID NOS: 41-44, 51-55 and 63-68.

Exemplary sequences of the invention are provided below in Tables 1-4.The header term “ID” refers to the given SEQ ID NO. Preferredcombinations of FR and CDR sequences for each NB construct are usedinterchangeably throughout the application.

TABLE 1  Polypeptide sequences of monovalent/monomeric DR5-bindingcomponents from initial screen, with SEQ ID NOs. Name Sequences ID 4E6evqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSV 1KGrftisrdnakntyymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvss 7A12evqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSV 2KGrftisrdnakntyylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvss 10F1evqlvesggglvqpggslrlscaasgftfsRYWMYwyrqapgkglewvsAINSGGGDTYYRDSV 3RGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaggtqvtvss 11D1evqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkgrelvaEITPRGRTNYADSEK 4SrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvss 11H6evqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVK 5GrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvss 11H6aevqllesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVK 87GrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvss

TABLE 2 Polypeptide sequences of multilalent anti-DR5 NB constructs with optionallinker sequences, with SEQ ID NOs. Name Sequence ID 4E6 trievqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADS 6VKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqg tqvtvss4E6 evqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADS 7tetra VKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvss 4E6evqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADS 8 pentaVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesgggsvgagdslrlscaasgrtfgSIRVGwfrqtpgkerefvaAINRNDGTTYYADSVKGrftisrdnakntvymqmaslkpedtavyycaaGLQYNRSADRVPVGAVYwgqgtqvtvss 7A12evqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDS 9 triVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvs s 7A12evqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDS 10 tetraVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvss 7A12evqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDS 11 pentaVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvgaggslrlscaasgrtfsNYAMGwfrqapgkerefvaALNWSGGSTYYVDSVKGrftisrdnakntvylqmnslkpedtavyycaaAGSFSLGGRPYGDDYwgkgtlv tvss10F1 evqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDS 12tri9 VRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsgggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsgggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvss 10F1evqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDS 13tri 20 VRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvss 10F1evqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDS 14 triVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqg tqvtvss10F1 evqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDS 15tetra VRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvss 10F1evqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDS 16 pentaVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgftfsRYWMYwvrqapgkglewvsAINSGGGDTYYRDSVRGrftisrdnfkntlylqmnslksedtavyycakAEGPPTFSLIRTMTVDPgaqgtqvtvss 11D1evqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSE 17 triKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvss 11D1evqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSE 18 tetraKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvss 11D1evqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSE 19 pentaKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgsidsINNMGwyrqapgkqrelvaEITPRGRTNYADSEKSrftisrdnakrtvnlqmnslkpedtavyycnaEVRERGTSWYRPDYwgqgtqvtvss 11H6evqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSV 20 triKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvss 11H6evqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSV 21 tetraKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfonaEILKRAYIDVYVNYwgqgtqvtvss 11H6evqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSV 22 pentaKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfonaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvss Unk1ggggsggggsggggsggggsggggsggggsggggs 23 Unk2 ggggsggggsggggsggggs 24Link 3 ggggsgggs 25 Link 4 GGGGS 81 Link 5 GGGS 82 11H6aevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSV 88tetra2 KGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqllesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqlvesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvssggggsggggsggggsggggsggggsggggsggggsevqllesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSVKGrftisrdnakdtmylqmnslkpedtavyfcnaEILKRAYIDVYVNYwgqgtqvtvss Tag1 EQKLISEEDLN 91 Tag2 HHHHHH 92 Tag3AAAEQKLISEEDLNGAAHHHHHH 93 Tag4 GAAEQKLISEEDLNGAAHHHHHH 94 Tag5 GGGC 95

TABLE 3  Human Optimized Nucleotide (NT) and Polypeptide (AA) SequencesName Sequence ID: 4E6 huevqllesggglvqpggslrlscaasgrtfgSIRVGwfrqapgkgrefvsAINRNDGTTYYADS 26 (AA)VKGrftisrdnskntvylqmnslrpedtavyycaaGLQYNRAADRVPVGAVYwgqgtlvtvss 4E6 huEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADS 27trimer VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSS(AA) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQG TLVTVSS4E6 hu EVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADS28 tetramerVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSS (AA)GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSS 4E6 huEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADS 29pentamer VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSS(AA) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVGWFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSS 4E6 hugaggtgcagctgctggagtctggcggcggactggtgcagcctggcggctccctgagactgtcctgcgccg96 Monomercctccggccggaccttcggctccatcagagtgggctggttccggcaggcccctggcaagggccgggagtt(NT)cgtgtccgccatcaaccggaacgacggcaccacctactacgccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccgtgtacctgcagatgaactccctgcggcccgaggacaccgccgtgtactactgcgccgctggcctgcagtacaacagagccgccgacagagtgcctgtgggcgctgtgtactggggccagggcaccctggtgaccgtgtcctct 4E6 hugaggtgcagctgctggagtctggcggcggactggtgcagcctggcggctccctgagactgtcctgcgccg97 Monomercctccggccggaccttcggctccatcagagtgggctggttccggcaggcccctggcaagggccgggagtt(NT)cgtgtccgccatcaaccggaacgacggcaccacctactacgccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccgtgtacctgcagatgaactccctgcggcccgaggacaccgccgtgtactactgcgccgctggcctgcagtacaacagagccgccgacagagtgcctgtgggcgctgtgtactggggccagggcaccctggtgaccgtgtcctctggcggcggaggatctggagggggaggaagcggcggaggaggatctggcggcggaggaagtgggggcggagggagtggcggaggtggaagtggtggagggggcagcgaggtgcagctgctggagagcggcggaggactggtgcagccaggcggatctctgcgcctgagctgcgccgccagcggcagaacctttggcagcatccgcgtgggatggttcagacaggctcccggaaagggacgcgagtttgtgtctgctatcaatcgcaatgatggcaccacatactatgctgatagcgtgaagggaagattcaccatcagccgcgacaatagcaagaatacagtgtatctgcagatgaatagcctgcgcccagaggatacagctgtgtattactgtgctgccggactgcagtataaccgggctgccgatcgggtgcccgtgggagccgtgtattggggacagggaacactggtgacagtgtcctctggcggcggaggatctgggggtggcggatctggcggcggaggaagcggtggcggaggatctggcggcggaggaagcggagggggaggatctggcggcggaggatctgaggtgcagctgctggagtccggcggaggactggtgcagccaggcggcagcctgcggctgtcttgcgccgcttctggcagaacattcggctctatccgcgtgggctggtttaggcaggctccaggcaagggacgcgagttcgtgagcgctatcaacagaaacgatggcacaacctattatgctgattctgtgaagggcaggtttacaatcagcagggataattctaagaataccgtgtacctgcagatgaactctctgaggccagaggataccgctgtgtactattgcgctgccggcctgcagtataatagggccgctgaccgcgtgccagtgggcgccgtgtattggggccagggcaccctggtgacagtgtcctctggcggaggtggcagcggcggtggcggatctggcggcggaggaagtgggggcggaggatctggcggcggaggaagcggcggagggggatctggcggcggaggatctgaggtgcagctgctggagtctggcggaggactggtgcagcctggcggaagcctgagactgagctgtgctgcttctggccgcaccttcggaagcatcagagtgggatggtttcgccaggctccaggaaagggccgggagttcgtctctgctatcaatagaaatgacggaacaacatattacgccgacagcgtgaagggacgctttacaatctctagggataacagcaagaacaccgtgtatctgcagatgaacagcctgcggcccgaggataccgccgtgtattattgtgccgctggactgcagtacaatcgggccgctgatagagtgcctgtgggagccgtgtactggggccagggcacactggtgacagtgtctagc 11H6 huevqlesggglvqpggslrlscaasgtfdkINNMGwyrqapgkqrdlvaQITPGGITDYADSV 30 (AA)KGrftisrdnskntlylqmnslrpedtavyycnaEILKRAYIDVYVNYwgqgtlvtvss 11H6 huEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSV 31trimer KGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGG(AA) SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSS 11H6 huEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSV 32tetramer KGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGG(AA) SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSS 11H6 huEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSV 33pentamer KGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGG(AA) SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMGWYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSS 11H6 hugaggtgcagctgctggagtctggcggcggactggtgcagcctggcggctccctgagactgtcctgcgccg98 monomercctccggcaccttcgacaagatcaacaacatgggctggtacaggcaggcccctggcaagcagagggacct(NT)ggtggcccagatcacccctggcggcatcaccgactacgccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccctgtacctgcagatgaactccctgcggcccgaggacaccgccgtgtactactgcaacgccgagatcctgaagcgggcctacatcgacgtgtacgtgaactactggggccagggcaccctggtgaccgtgtcctct 11H6 hugaggtgcagctgctggagtctggcggcggactggtgcagcctggcggctccctgagactgtcctgcgccg99 tetramercctccggcaccttcgacaagatcaacaacatgggctggtacaggcaggcccctggcaagcagagggacct(NT)ggtggcccagatcacccctggcggcatcaccgactacgccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccctgtacctgcagatgaactccctgcggcccgaggacaccgccgtgtactactgcaacgccgagatcctgaagcgggcctacatcgacgtgtacgtgaactactggggccagggcaccctggtgaccgtgtcctctggcggcggaggatctggagggggaggaagcggcggaggaggatctggcggcggaggaagtgggggcggagggagtggcggaggtggaagtggtggagggggcagcgaggtgcagctgctggagagcggcggaggactggtgcagccaggcggatctctgcgcctgagctgcgccgccagcggcacatttgataagatcaataatatgggatggtatcgccaggctccaggcaagcagcgcgatctggtggctcagatcacaccaggcggaatcacagattatgccgatagcgtgaagggaagattcaccatcagccgcgacaatagcaagaatacactgtatctgcagatgaatagcctgcgcccagaggatacagctgtgtattactgtaatgctgagatcctgaagcgcgcttatatcgatgtgtatgtgaattattggggacagggaacactggtgacagtgtcctctggcggcggaggatctgggggtggcggatctggcggcggaggaagcggtggcggaggatctggcggcggaggaagcggagggggaggatctggcggcggaggatctgaggtgcagctgctggagtccggcggaggactggtgcagccaggcggcagcctgcggctgtcttgcgccgcttctggcaccttcgataagatcaacaatatgggatggtacagacaggctcccggaaagcagcgggatctggtggcccagatcaccccaggcggcatcacagattacgctgattctgtgaagggcaggtttacaatcagcagggataattctaagaataccctgtacctgcagatgaactctctgaggccagaggataccgctgtgtactattgtaacgccgagatcctgaagagggcttacatcgatgtgtacgtgaattattggggccagggcaccctggtgacagtgtcctctggcggaggtggcagcggcggtggcggatctggcggcggaggaagtgggggcggaggatctggcggcggaggaagcggcggagggggatctggcggcggaggatctgaggtgcagctgctggagtctggcggaggactggtgcagcctggcggaagcctgagactgagctgtgctgcttctggcaccttcgacaagatcaataatatgggctggtatagacaggccccaggaaagcagagggacctggtcgctcagatcacacccggcggaatcaccgactacgctgacagcgtgaagggacgctttacaatctctagggataacagcaagaacaccctgtatctgcagatgaacagcctgcggcccgaggataccgccgtgtattattgcaatgctgagatcctgaagagggcctatatcgacgtgtatgtgaattactggggccagggcacactggtgacagtgtcctct

TABLE 4 Sequences for CDRs and frameworks, plus preferred combinations asprovided in for formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 CloneFR1 ID CDR1 ID FR2 ID 7A12 evqlvesggglvqaggslrlscaasgrtfs 34 NYAMG 41wfrqapgkerefva 45 4E6 hu evqllesggglvqpggslrlscaasgrtfg 35 SIRVG 42wfrqapgkgrefvs 46 4E6 evqlvesgggsvgagdslrlscaasgrtfg 36 SIRVG 42wfrqtpgkerefva 47 11H6 hu evqllesggglvqpggslrlscaasgtfdk 37 INNMG 43wyrqapgkqrdlva 48 11H6 evqlvesggglvqpggslrlscaasgtfdk 38 INNMG 43wyrqapgkqrdlva 48 11D1 evqlvesggglvqpggslrlscaasgsids 39 INNMG 43wyrqapgkqrelva 49 10F1 evqlvesggglvqpggslrlscaasgftfs 40 RYWMY 44wvrqapgkglewvs 50 Clone CDR2 ID FR3 ID 7A12 ALNWSGGSTYYVDSVKG 51rftisrdnakntvylqmnslkpedtavyycaa 56 4E6 hu AINRNDGTTYYADSVKG 52rftisrdnskntvylqmnslrpedtavyycaa 57 4E6 AINRNDGTTYYADSVKG 52rftisrdnakntvymqmaslkpedtavyycaa 58 11H6 hu QITPGGITDYADSVKG 53rftisrdnskntlylqmnslrpedtavyycna 59 11H6 QITPGGITDYADSVKG 53rftisrdnakdtmylqmnslkpedtavyfcna 60 11D1 EITPRGRTNYADSEKS 54rftisrdnakrtvnlqmnslkpedtavyycna 61 10F1 AINSGGGDTYYRDSVRG 55rftisrdnfkntlylqmnslksedtavyycak 62 Clone CDR3 ID FR4 ID 7A12AGSFSLGGRPYGDDY 63 wgkgtlvtvss 69 4E6 hu GLQYNRAADRVPVGAVY 64wgqgtlvtvss 70 4E6 GLQYNRSADRVPVGAVY 65 wgqgtqvtvss 71 11H6 huEILKRAYIDVYVNY 66 wgqgtlvtvss 70 11H6 EILKRAYIDVYVNY 66 wgqgtqvtvss 7111D1 EVRERGTSWYRPDY 67 wgqgtqvtvss 71 10F1 AEGPPTFSLIRTMTVDP 68gaqgtqvtvss 72

Further aspects of the NB agents of the invention are provided below.

The compounds, (monovalent or multivalent) polypeptides and compositionsdescribed herein may be such that they bind DR5 but not to the bindingsite of TRAIL on the DR5, and/or may be such that they do not competewith TRAIL for binding to the DR5.

Such compounds and polypeptides may be such that their binding to theDR5 induces, triggers, increases or enhances the signaling mediated bythe DR5, and in particular triggers or induces apoptosis in the cell onwhich the DR5 is present. Such compounds and polypeptides may also besuch that they increase or enhance the signaling that is mediated byTRAIL and the DR5 upon binding of TRAIL to its receptor. According tothis aspect, binding of both TRAIL as well as the compounds/polypeptidesdescribed herein to the same receptor may lead to a synergistic effecton such signaling.

In one specific aspect, the compounds and polypeptides described hereinare multimeric compounds and polypeptides (as described herein) directedagainst DR5. In particular, the compounds and polypeptides describedherein may be multimeric polypeptides that are capable of multimerizingDR5 on a cell membrane and as such are capable of inducing, triggering,increasing or enhancing the signaling mediated by the DR5, and more inparticular of triggering or inducing apoptosis in the cell on which theDR5 is present. In one embodiment, such monomeric or multimericcompounds or polypeptides disclosed herein may bind DR5 at or near thebinding site of TRAIL on the DR5, and/or may compete with TRAIL forbinding to DR5. In one embodiment, they do not bind to the binding sitefor TRAIL on the DR5, essentially do not hinder or prevent binding ofTRAIL to the DR5, and/or do not compete with TRAIL for binding to theDR5, and even be such that they increase or enhance the signaling thatis mediated by TRAIL and the DR5 upon binding of TRAIL to its receptor(which may again result in a synergistic effect upon the signalingmediated by the DR5). In one embodiment, the amino acid sequencecontains a portion derived from naturally occurring TRAIL ligand, i.e.,it comprises a segment made up of a natural TRAIL ligand polypeptide orany TRAIL-receptor binding fragment of such TRAIL-ligand.

The polypeptides and compositions of the present invention can be usedfor the prevention and treatment of diseases and disorders associatedwith DR5. Specifically, “diseases and disorders associated with DR5” aredefined as those that can be prevented and/or treated, respectively, bysuitably administering to a subject in need thereof (i.e., having thedisease or disorder or at least one symptom thereof and/or at risk ofattracting or developing the disease or disorder) of either apolypeptide or composition of the invention (and in particular, of apharmaceutically active amount thereof) and/or of a known activeprinciple active against DR5 or a biological pathway or mechanism inwhich DR5 is involved (and in particular, of a pharmaceutically activeamount thereof), which results in the modulation, i.e., agonism orantagonism, of DR5-mediated signaling. As listed below, diseases anddisorders treatable by the induction of apoptosis in the cell or tissuetargeted in the disease are particularly included herein as beingassociated with DR5.

Diseases and disorders associated with DR5 may in particular be diseasesand disorders that can be treated by triggering, initiating, increasingor enhancing the signaling, mechanisms, responses and effects in which aDR5 is involved (in other words, by effecting an agonistic effect on aDR5 or on DR5 mediated signaling). More in particular, diseases anddisorders associated with DR5 may be diseases and disorders that can betreated by triggering, initiating, increasing or enhancing cellapoptosis in one or more cells or tissues in the subject to be treated.

In one embodiment, the NB agents of the invention are agonists of theTRAIL receptors DR5, having the potential to be used in therapy againsta wide range of diseases associated with DR5. Examples of diseases anddisorders associated with DR5 will be clear to the skilled person basedon the disclosure herein. In one embodiment, inventive NB agents haveutility in the treatment of proliferative diseases including, e.g.,cancers such as solid tumors, primary and metastatic cancers such asrenal cell carcinoma, and cancers of the lung (e.g., small cell lungcancer “SCLC” and non-small cell lung cancer “NSCLC”), pancreas,hematopoietic malignancy, glioma, astrocytoma, mesothelioma, colorectalcancers, prostate cancer, osteosarcoma, melanoma, lymphoma (includingbut not limited to Burkitt's Lymphoma), breast cancer, endometrialcancer, liver cancer, gastric cancer, skin cancer, ovarian cancer andsquamous cell cancers of any origin (e.g., lung, head and neck, breast,thyroid, cervix, skin, esophageal, etc.), as well as liquid cancers,e.g., such as leukemias including especially a T-cell leukemia such asacute T-cell leukemia (T-ALL), acute B-cell leukemia (B-ALL), chronicmyelogenous leukemia (CML), acute myelogenous leukemia (AML), plasmacell myeloma and multiple myeloma (MM). In one embodiment, the inventiveNB agents have utility in the treatment of non-cancer indicationsincluding, but not limited to, e.g., inflammatory and autoimmunediseases, such as systemic lupus erythematosus, Hashimoto's disease,rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome,pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome,Crohn's disease, autoimmune hemolytic anemia, sterility, myastheniagravis, multiple sclerosis, Basedow's disease, thromboticthrobocytopenia, thrombopenia purpurea, insulin-dependent diabetesmellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; globerula nephritis; and hypoplasticanemia.

Thus, without being limited thereto, the amino acid sequences andpolypeptides of the invention can, e.g., be used to prevent and/or totreat all diseases and disorders that are currently being prevented ortreated with active principles that can modulate DR5-mediated signaling,such as those mentioned herein and in the art references cited. In oneembodiment the polypeptides of the invention can be used to preventand/or to treat all diseases and disorders for which treatment with suchactive principles is currently being developed, has been proposed, orwill be proposed or developed in future. In one embodiment, it isenvisaged that, because of their favorable properties as furtherdescribed herein, the polypeptides herein may be used for the preventionand treatment of other diseases and disorders than those for which theseknown active principles are being used or will be proposed or developed;and/or that the polypeptides of the present invention may provide newmethods and regimens for treating the diseases and disorders describedherein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

In one embodiment, the invention provides pharmacologically activeagents, as well as compositions comprising the same (whether or notcomplete polypeptides as described herein, and/or fragments and/ormultimeric variants of said polypeptides or fragment, all of which arebroadly referred to herein as “inventive compositions”), that can beused in the diagnosis, prevention and/or treatment of diseases anddisorders associated with DR5 and of the further diseases and disordersmentioned herein; and to provide methods for the diagnosis, preventionand/or treatment of such diseases and disorders that involve theadministration and/or use of such agents and compositions.

In one embodiment the invention provides such pharmacologically activeagents, compositions and/or methods that have certain advantagescompared to the agents, compositions and/or methods that are currentlyused and/or known in the art. These advantages will become clear fromthe further description below.

In one embodiment, the invention provides amino acid sequences that aredirected against DR5, in particular against DR5 from a warm-bloodedanimal, more in particular against DR5 from a mammal, and especiallyagainst human DR5; and to provide proteins and polypeptides comprisingor essentially consisting of at least one such amino acid sequence.

In one embodiment the invention provides such amino acid sequences andsuch proteins and/or polypeptides that are suitable for prophylactic,therapeutic and/or diagnostic use in a warm-blooded animal, and inparticular in a mammal, and especially in a human being.

In one embodiment the invention provides such amino acid sequences andsuch proteins and/or polypeptides that can be used for the prevention,treatment, alleviation and/or diagnosis of one or more diseases,disorders or conditions associated with DR5 and/or mediated by DR5 (suchas the diseases, disorders and conditions mentioned herein) in awarm-blooded animal, in particular in a mammal, and more in particularin a human being.

In one embodiment the invention provides such inventive compositionsthat for use in the preparation of pharmaceutical or veterinarycompositions for the prevention and/or treatment of one or morediseases, disorders or conditions associated with and/or mediated by DR5(such as the diseases, disorders and conditions mentioned herein) in awarm-blooded animal, in particular in a mammal, and more in particularin a human being.

In one embodiment, the invention provides inventive compositions thatare directed against and/or can specifically bind to DR5; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence. More specifically,the invention provides amino acid sequences that can specifically bindto DR5 cell surface receptor as e.g., defined with the accession number“BAA33723” to be found at e.g. the NCBI protein database. In particular,the invention provides amino acid sequences that can specifically bindto DR5 cell surface receptor as e.g. defined with the accession number“BAA33723” to be found at e.g. the NCBI protein database and that doesnot bind to at least another TRAIL-receptor, such as TRAIL-R3 andTRAIL-R4. A short from splice variant of human DR5 is also known(GenBank entry NP_(—)671716).

In one embodiment, the invention provides amino acid sequences that canbind to DR5 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, inthe pharmaceutically acceptable range as provided herein; as well ascompounds and constructs, and in particular proteins and polypeptides,that comprise at least one such amino acid sequence.

In one embodiment, inventive compositions are such that they:

a) bind to DR5 with a dissociation constant (K_(D)) of 10⁻⁶ to 10⁻¹²moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or lessand more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e., with an associationconstant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²liter/moles); and/or

b) bind to DR5 with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷M⁻¹s⁻¹ preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferablybetween 10⁴ M⁻¹ s and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷M⁻¹s⁻¹; and/or

-   -   c) bind to DR5 with a k_(off) rate between 1 s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

In one embodiment, an inventive composition, whether monovalent ormultivalent, binds to DR5 with an affinity less than 100 nM, and/or lessthan 10 nM, and/or less than 1 nM, such as less than 500 pM. In oneembodiment, the inventive composition is a tetravalent composition. Inone embodiment, the inventive composition is a pentavalent composition.

In one embodiment, an inventive composition, whether monovalent ormultivalent, will specifically induce apoptosis in various cancer celltypes (e.g., at least one type) with an IC₅₀ less than 100 nM,preferably less than 10 nM, more preferably less than 1 nM, such as lessthan 500 pM. In one embodiment, a multivalent amino acid sequence of theinvention is at least 10 fold more potent in tumor cell line than innormal (non-tumor) cell line, or at least 100 fold more potent than innormal (non-tumor) cell line, as measured, e.g., in non-tumor cell linesmentioned in the experimental part, e.g. Huvec, IMR-90 and ARPE-19. Seeexamples and Table 13.

For binding to DR5, an inventive composition will usually contain withinits amino acid sequence one or more amino acid residues or one or morestretches of amino acid residues (i.e., with each “stretch” comprisingtwo or more amino acid residues that are adjacent to each other or inclose proximity to each other, i.e., in the primary (linear) or tertiary(conformational) structure of the amino acid sequence) via which theamino acid sequence of the invention can bind to DR5, which amino acidresidues or stretches of amino acid residues thus form the “site” forbinding to DR5 (also referred to herein as the “antigen binding site” or“epitope”). Epitope mapping experiments are provided herein in theExamples.

In one embodiment the inventive compositions provided herein are inessentially isolated form, or form part of a protein or polypeptide ofthe invention that may comprise or essentially consist of one or moreamino acid sequences herein disclosed. In one embodiment the inventivecompositions may further comprise one or more additional amino acidsequences. In one embodiment, such additional sequences are linked viaone or more suitable linkers. Without limitation, the inventivecompositions may be used as a binding unit in such a protein orpolypeptide. Such inventive compositions may contain one or more furtheramino acid sequences that can serve as an additional binding unit (i.e.,against one or more other targets than DR5), so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively. Such a protein or polypeptide may also be in essentiallyisolated form.

In one embodiment the inventive compositions essentially consist of asingle amino acid chain that is not linked via disulphide bridges to anyother amino acid sequence or chain (but that may or may not contain oneor more intramolecular disulphide bridges) or pharmaceutically relevantcomposition. As an example of an intramolecular disulfide bridge, it isknown that camelid V_(HH) constructs may sometimes contain a disulphidebridge between CDR3 and CDR1 or FR2. In one embodiment, inventivecompositions may be linked to each other and/or to other amino acidsequences (e.g. via disulphide bridges) to provide alternative peptideconstructs (for example Fab′ fragments, F(ab′)₂ fragments, ScFvconstructs, “diabodies” and other multivalent and/or multispecificconstructs. See review by Holliger and Hudson, Nat Biotechnol. 2005September; 23(9): 1126-36). Examples of pharmaceutically relevantcompositions include drugs that treat a disease, or compositions thatincrease half life, target specific tissues, and/or kill the targetcell.

In one embodiment the inventive compositions essentially consist of asingle amino acid chain that is linked via at least one intermoleculardisulphide bridge to any other amino acid sequence or chain (either ofwhich may or may not contain one or more intramolecular disulphidebridges) or pharmaceutically relevant composition.

In one embodiment, when an inventive composition is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is either an amino acidsequence that does not occur naturally in said subject; or, when it doesoccur naturally in said subject, is in essentially isolated form.

In one embodiment for pharmaceutical use, the disclosed amino acidsequences of the invention (as well as compounds, constructs andpolypeptides comprising the same) are directed against human DR5. In oneembodiment for veterinary purposes, such disclosed compositions aredirected against DR5 from the species to be treated. In one embodimentfor veterinary purposes, such disclosed compositions are cross-reactivewith DR5 from the species to be treated.

In one embodiment, an inventive amino acid sequence has at least onebinding site for binding against DR5, and may contain one or morefurther binding sites for binding against other epitopes, antigens,proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. These may, e.g., be assays ormodels for measuring the influence of the amino acid sequences orcompounds described herein on apoptosis, on the proliferation of tumorcells, and/or on the growth of tumors in an animal model for such atumor. Suitable assays and animal models will be clear to the skilledperson, and, e.g., include assays and animal models used in theexperimental part below and in the art references cited herein. Examplesare binding assays such as ELISA, FACS or surface plasmon resonancemethodology; functional in vitro assays such as in vitro cell survivalassay with a panel of tumor and normal cell lines, in vitro efficacyassay with Jurkat cell survival detection; functional in vivo assayssuch as single dose in vivo study combined with a caspase 3/7 activationread out providing various efficacy, PK and PD data.

In one embodiment according to the invention, amino acid sequences andpolypeptides that are directed against DR5 from a first species ofwarm-blooded animal may or may not show cross-reactivity with DR5 fromone or more other species of warm-blooded animal. For example, aminoacid sequences and polypeptides directed against human DR5 may or maynot show cross reactivity with DR5 from one or more other species ofprimates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomologus monkeys (Macaca fascicularis,a.k.a., “cyno”) and/or rhesus monkeys (Macaca mulatta)) and baboon(Papio ursinus)) and/or with DR5 from one or more species of animalsthat are often used in animal models for diseases (for example mouse,rat, rabbit, pig or dog), and in particular in animal models fordiseases and disorders associated with DR5 (such as the species andanimal models mentioned herein). In this respect, it will be clear tothe skilled person that such cross-reactivity, when present, may haveadvantages from a drug development point of view, since it allows theamino acid sequences and polypeptides against human DR5 to be tested insuch disease models. Various inventive constructs provided herein showspecific cross-reactivity with cyno DR5.

In one embodiment, amino acid sequences and polypeptides of theinvention that are cross-reactive with DR5 from multiple species ofmammal will usually be advantageous for use in veterinary applications,since it will allow the same amino acid sequence or polypeptide to beused across multiple species. Thus, in one embodiment amino acidsequences and polypeptides directed against DR5 from one species ofanimal (such as amino acid sequences and polypeptides against human DR5)can be used in the treatment of another species of animal, as long asthe use of the amino acid sequences and/or polypeptides provide thedesired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of DR5 againstwhich the amino acid sequences and polypeptides of the invention aredirected. However, in one embodiment the inventive amino acid sequencesand polypeptides are directed against the extracellular domain of theDR5. In one non-limiting aspect, the inventive amino acid sequences andpolypeptides are directed against the DR5-binding domain of DR5, and areas further defined herein. In one embodiment, a monovalent ormultivalent amino acid sequence and polypeptides of the inventioncompete with the natural ligand of the DR5 in a competitive bindingassay as described in the experimental part. In one embodiment, amonovalent or multivalent amino acid sequence and polypeptides of theinvention binds synergistically with the natural ligand of the DR5 in acompetitive binding assay.

In one embodiment of the invention, where applicable, an amino acidsequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofDR5. In such a case, the antigenic determinants, epitopes, parts,domains or subunits of DR5 to which the amino acid sequences and/orpolypeptides of the invention bind may be essentially the same (forexample, if DR5 contains repeated structural motifs or occurs in amultimeric form) or may be different (and in the latter case, the aminoacid sequences and polypeptides of the invention may bind to suchdifferent antigenic determinants, epitopes, parts, domains, subunits ofDR5 with an affinity and/or specificity which may be the same ordifferent). Also, e.g., when DR5 exists in an activated conformation andin an inactive conformation, the amino acid sequences and polypeptidesof the invention may bind to either one of these confirmation, or maybind to both these confirmations (i.e., with an affinity and/orspecificity which may be the same or different). Also, e.g., the aminoacid sequences and polypeptides of the invention may bind to aconformation of DR5 in which it is bound to a pertinent ligand, may bindto a conformation of DR5 in which it not bound to a pertinent ligand, ormay bind to both such conformations (again with an affinity and/orspecificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofDR5; or at least to those analogs, variants, mutants, alleles, parts andfragments of DR5 that contain one or more antigenic determinants orepitopes that are essentially the same as the antigenic determinant(s)or epitope(s) to which the amino acid sequences and polypeptides of theinvention bind in DR5 (e.g. in wild-type DR5). Again, in such a case,the amino acid sequences and polypeptides of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e., higher than or lower than), the affinity and specificitywith which the amino acid sequences of the invention bind to (wild-type)DR5. Also included within the inventive scope is that the amino acidsequences and polypeptides of the invention bind to some analogs,variants, mutants, alleles, parts and fragments of DR5, but not toothers.

In general, when DR5 exists in a monomeric form and in one or moremultimeric forms, it is within the scope of the invention that the aminoacid sequences and polypeptides of the invention only bind to DR5 inmonomeric form, only bind to DR5 in multimeric form, or bind to both themonomeric and the multimeric form. Again, in such a case, the amino acidsequences and polypeptides of the invention may bind to the monomericform with an affinity and/or specificity that are the same as, or thatare different from (i.e., higher than or lower than), the affinity andspecificity with which the amino acid sequences of the invention bind tothe multimeric form.

Also, when DR5 can associate with other proteins or polypeptides to formprotein complexes (e.g. with multiple subunits), it is within the scopeof the invention that the amino acid sequences and polypeptides of theinvention bind to DR5 in its non-associated state, bind to DR5 in itsassociated state, or bind to both. In all these cases, the amino acidsequences and polypeptides of the invention may bind to such multimersor associated protein complexes with an affinity and/or specificity thatmay be the same as or different from (i.e., higher than or lower than)the affinity and/or specificity with which the amino acid sequences andpolypeptides of the invention bind to DR5 in its monomeric andnon-associated state.

In one embodiment, as will be clear to the skilled person, inventivecompositions that contain two or more amino acid sequences directedagainst DR5 may bind with higher avidity to DR5 than the correspondingmonomeric amino acid sequence(s). For example, and without limitation,proteins or polypeptides that contain two, three, four, five, six,eight, ten or more operably linked monomeric units of amino acidsequences directed against the same or different epitopes of DR5 may(and usually will) bind with higher avidity than each of the differentmonomers, and proteins or polypeptides that contain two, three, four ormore amino acid sequences directed against DR5 may (and usually will)bind also with higher avidity to a multimer of DR5. In one embodiment,the inventive composition is a single polypeptide chain made of fourmonomers directed against DR5 connected by amino acid linkers. In oneembodiment, the inventive composition is a single polypeptide chain madeof five monomers directed against DR5 connected by amino acid linkers.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of DR5 (including monomeric, multimeric andassociated forms) that are the most relevant from a biological and/ortherapeutic point of view, as will be clear to the skilled person.

It is within the scope of the invention to use compositions comprisingparts, fragments, analogs, mutants, variants, alleles and/or derivativesof the amino acid sequences and polypeptides of the invention, and/or touse proteins or polypeptides comprising or essentially consisting of oneor more of such compositions as long as these are suitable for the usesenvisaged herein. In one embodiment such compositions will contain (atleast part of) a functional antigen-binding site for binding againstDR5. In one embodiment such compositions will be capable of specificbinding to DR5. In one embodiment such compositions will be capable ofbinding to DR5 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as provided herein. Some non-limitingexamples of such compositions will become clear from the furtherdescription herein. Additional fragments or polypeptides of theinvention may also be provided by suitably combining (i.e., by linkingor genetic fusion) one or more (smaller) compositions as describedherein.

In one non-limiting aspect of the invention, analogs, mutants, variants,alleles, and/or derivatives of the polypeptides of the invention have anincreased half-life in serum (as further described herein) compared tothe amino acid sequence from which they have been derived. For example,an amino acid sequence of the invention may be linked (chemically orotherwise) to one or more groups or moieties that extend the half-life(such as PEG), so as to provide a derivative of an amino acid sequenceof the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e., by folding). Reference is made inter alia tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. In oneembodiment, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding to DR5. Inone further embodiment, such a construct is capable of binding to DR5with an affinity (suitably measured and/or expressed as a K_(D)-value(actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rateand/or a k_(off)-rate, or alternatively as an IC₅₀ value, as furtherdescribed herein) that is as provided herein. In one embodiment, parts,fragments, analogs, mutants, variants, alleles and/or derivatives ofsuch amino acid sequences are such that they comprise an immunoglobulinfold or are capable for forming, under suitable conditions, animmunoglobulin fold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of fourframework regions (FR1, FR2, FR3 and FR4 respectively) and threeinterspersed complementarity determining regions (CDR1, CDR2 and CDR3respectively); or any suitable fragment of such an amino acid sequence(which will then usually contain at least some of the amino acidresidues that form at least one of the CDR's, as further describedherein). In one embodiment these regions are arranged as shown informula I:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (formula I)

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin variable domain sequence or a suitablefragment thereof, such as light chain variable domain sequence (e.g. aV_(L) sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H) sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence that is derived from aso-called “heavy chain antibody”.

It should be noted that the invention is not limited as to the origin ofthe amino acid sequence of the invention (or of the nucleotide sequenceof the invention used to express it), nor as to the way that the aminoacid sequence or nucleotide sequence of the invention is (or has been)generated or obtained. Thus, the amino acid sequences of the inventionmay contain naturally occurring amino acid sequences (from any suitablespecies) or synthetic or semi-synthetic amino acid sequences. In aspecific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” immunoglobulinsequences (e.g., such as partially or fully humanized mouse, rabbit ormonkey immunoglobulin sequences (or immunoglobulin sequences of anyother mammalian species contemplated by one skilled in the art), and inparticular partially or fully humanized camelid V_(HH) sequences orfragments thereof), “camelized” immunoglobulin sequences, as well asimmunoglobulin sequences that have been obtained by techniques such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, veneering,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the above. Reference is, e.g.,made to standard handbooks, as well as to the further description andart references mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may, e.g., be sequences that are isolated by PCR from a suitablenaturally occurring template (e.g. DNA or RNA isolated from a cell),nucleotide sequences that have been isolated from a library (and inparticular, an expression library), nucleotide sequences that have beenprepared by introducing mutations into a naturally occurring nucleotidesequence (using any suitable technique known per se, such as mismatchPCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domainantibody (or an amino acid sequence that is suitable for use as a domainantibody), a single domain antibody (or an amino acid sequence that issuitable for use as a single domain antibody), a “dAb” (or an amino acidsequence that is suitable for use as a dAb) or a camelid antibody (andincluding but not limited to a V_(HH) sequence or variants thereof);other single variable domains, or any suitable fragment of any onethereof. For a general description of (single) domain antibodies,reference is also made to the art references cited above, as well as toEP 0 368 684. For the term “dAb's”, reference is, e.g., made to Ward etal. (Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., TrendsBiotechnol., 2003, 21(11): 484-490; as well as to, e.g., WO 06/030220,WO 06/003388 and other published patent applications of Domantis Ltd. Inone embodiment of the invention, single domain antibodies or singlevariable domains can be derived from certain species of shark (forexample, the so-called “IgNAR domains”, see e.g., WO 05/18629).

In particular, the amino acid sequence of the invention may be a camelidheavy chain variable domain construct (e.g., a V_(HH) or variablesthereof, including variables optimized for use as a human therapeutic,e.g., as defined in, e.g., WO 2008/020079) or a suitable fragment orvariant thereof. In one embodiment, the amino acid sequence is aNANOBODY® or a construct thereof. Note that NANOBODY™, NANOBODIES™ andNANOCLONE™ are registered trademarks of Ablynx N.V. Specific heavy chainvariable domain constructs directed against DR5, whether or not“humanized” or otherwise altered or optimized, will be referred toherein as a “NB Sequence” of the invention.

As used herein, the phrase “sequence optimized”, or alternativelysequences that are “altered or optimized” is defined to refer to thereplacement of residues of a NB agent, especially in the frameworkregion, to reduce the occurrence of an immunogenic response to the NBagent by the subject to which it is administered. In the case where thesubject is a human, the phrase includes any technique known in the artto humanize the sequence.

In one embodiment, NB agents are so-called “V_(H)3 class” (i.e., a NBconstruct with a high degree of sequence homology to human germlinesequences of the V_(H)3 class such as DP-47, DP-51 or DP-29). In oneembodiment, this V_(H)3 class of NB constructs comprise the frameworkregions of this invention. However, the invention in its broadest sensegenerally covers any type of NB agents directed against DR5. Forexample, in one embodiment, the invention also covers the NB constructsbelonging to the so-called “V_(H)4 class” (i.e., NB constructs with ahigh degree of sequence homology to human germline sequences of theV_(H)4 class such as DP-78). See e.g., WO 07/118670.

In one embodiment, NB constructs (in particular fully humanized andpartially humanized constructs) are characterized by the presence of oneor more “Hallmark residues” in one or more of the framework sequences.In one embodiment, the framework sequences present in the NB constructsof the invention may be such that the amino acid sequence is a variantof a camelid V_(HH) construct. Some non-limiting examples of (suitablecombinations of) such framework sequences and alternative Hallmarkresidues appear, e.g., in WO 2008/020079, pages 65-98, which pages areincorporated by reference in their entirety. Other humanized orpartially humanized residues known in the art are also contemplated asencompassed within the invention.

Generally, a V_(H) or a V_(HH) construct can be defined as an amino acidsequence with the (general) structure of formula I, in which FR1 to FR4refer to framework regions 1 to 4, respectively, and in which CDR1 toCDR3 refer to the complementarity determining regions 1 to 3,respectively.

In one embodiment an inventive composition may be a NB construct with anamino acid sequence with the (general) structure of formula I in whichone or more of the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108 according to the Kabat numbering are chosenfrom the Hallmark residues mentioned in Table A-3 in WO 2008/020079 andin which said amino acid sequence has at least 80%, 85%, 90%, 95%, 98%,99% or 100% amino acid identity with at least one of the framework aminoacid sequences of SEQ ID NO's: 1 to 22 in WO 2008/020079, in which forthe purposes of determining the degree of amino acid identity, the aminoacid residues that form the CDR sequences (indicated with X in thesequences) are disregarded.

In the NB agents of the invention, including without limitation thevarious formats or scaffolds, the CDR sequences are generally asprovided in Tables 1-3 and especially as specifically designated by SEQID NO in Table 4. In particular, CDR1 may be any one of SEQ ID NOs: 41to 44; CDR2 may be any one of SEQ ID NOs: 51 to 55; and CDR3 may be anyone of SEQ ID NOs: 63 to 68.

Thus, in one embodiment the invention relates to such V_(H) constructsthat can bind to and/or are directed against DR5, to suitable fragmentsthereof, as well as to polypeptides that comprise or essentially consistof one or more of such V_(H) constructs and/or suitable fragments.

In general, SEQ ID NOs: 1-22, 26-40, 87-88, and 102-103 provide theamino acid sequences of a number of NB Agents that have been raisedagainst DR5 (see Tables 1-4). SEQ ID NOs: 96-99 provide specific nucleicacid sequences that encode specific NB Agents as provided herein.

In some embodiments, inventive compositions are NB agents, e.g., asshown in Tables 1 through 4, that can bind to and/or are directedagainst to DR5 and that have at least 90% amino acid identity with atleast one of the amino acid sequences of SEQ ID NOs: 1-22, 26-40, 87-88and 102-103, in which for the purposes of determining the degree ofamino acid identity, the amino acid residues that form the CDR sequencesare disregarded and in which one or more of the amino acid residues atpositions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to theKabat numbering are Hallmark residues.

In various embodiments and as provided in Table 4, the NB constructscomprise framework 1 sequences of SEQ ID NO's: 34 to 40), framework 2sequences of SEQ ID NO's: 45 to 50, framework 3 sequences of SEQ IDNO's: 56 to 62 and framework 4 sequences of SEQ ID NO's: 69 to 72. Inone embodiment, amino acid residues at positions 1 to 4 and 27 to 30 ofthe framework 1 sequences of NB constructs of SEQ ID NO's: 1-22, 26-40,87-88 and 102-103 are disregarded for determining the degree of aminoacid identity.

In one embodiment, inventive NB agents and compositions may be derivedin any suitable manner and from any suitable source, and may, e.g., benaturally occurring V_(HH) sequences (i.e., from a suitable species ofCamelid) or synthetic or semi-synthetic amino acid sequences, includingbut not limited to “humanized” NB agents, “camelized” immunoglobulinsequences (and in particular camelized heavy chain variable domainsequences), as well as NB agents that have been obtained by techniquessuch as affinity maturation (for example, starting from synthetic,random or naturally occurring immunoglobulin sequences), CDR grafting,veneering, combining fragments derived from different immunoglobulinsequences, PCR assembly using overlapping primers, and similartechniques for engineering immunoglobulin sequences well known to theskilled person; or any suitable combination of any of the foregoing asfurther described herein. Also, when an inventive composition comprisesa V_(HH) sequence, said composition may be suitably humanized, asdescribed herein, so as to provide one or more further (partially orfully) humanized composition of the invention. In one embodiment, whenan inventive composition comprises a synthetic or semi-syntheticsequence (such as a partially humanized sequence), said composition mayoptionally be further suitably humanized, e.g., again either partiallyor fully humanized.

In one embodiment, humanized compositions are amino acid sequences thathave at least one amino acid residue present (and in particular, in atleast one of the framework residues) that is and/or that corresponds toa humanizing substitution. In such a non-limiting embodiment, humanizingsubstitutions (and suitable combinations thereof) will be clear to theskilled person based on the disclosure herein. In one embodiment,potentially useful humanizing substitutions can be ascertained bycomparing the sequence of the framework regions of a naturally occurringV_(HH) sequence with the corresponding framework sequence of one or moreclosely related human V_(H) sequences, after which one or more of thepotentially useful humanizing substitutions (or combinations thereof)thus determined can be introduced into said V_(HH) sequence in anymanner known per se. In one embodiment, the resulting humanized V_(HH)sequences are tested for affinity for the target, for stability, forease and level of expression, and/or for other desired properties. Inthis way, by means of a limited degree of trial and error, othersuitable humanizing substitutions (or suitable combinations thereof) canbe determined by the skilled person based on the disclosure herein.Also, based on the foregoing, (the framework regions of) an inventivecomposition of the invention may be partially humanized or fullyhumanized.

In one embodiment, humanized compositions herein are humanized variantsof the compositions of SEQ ID NOs: 1-22, 26-40, 87-88, and 102-103 andof SEQ ID NOS: 96-99. In one embodiment, humanized NB constructs are asprovided in Table 3.

Thus, some other preferred compositions of the invention arecompositions which can bind (as further defined herein) to DR5 and which(a) are a humanized variant of one of the amino acid sequences of SEQ IDNO's: 1-22, 26-40, 87-88, and 102-103; and/or (b) have at least 90%amino acid identity with at least one of the amino acid sequences of SEQID NO's: 1-22, 26-40, 87-88, and 102-103. In one embodiment, for thepurposes of determining the degree of amino acid identity, the aminoacid residues that form the CDR sequences are disregarded.

In one embodiment, one or more of the amino acid residues at positions11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabatnumbering are chosen from the Hallmark residues mentioned in Table A-3in WO 2008/020079.

In one embodiment, the invention provides a number of stretches of aminoacid residues (i.e., small peptides) that are particularly suited forbinding to DR5. These stretches of amino acid residues may be presentin, and/or may be incorporated into, an amino acid sequence of theinvention, in particular in such a way that they form (part of) theantigen binding site of an amino acid sequence of the invention. Asthese stretches of amino acid residues are first generated as CDRsequences of heavy chain antibodies or V_(HH) sequences that are raisedagainst DR5 (or may be based on and/or derived from such CDR sequences,as further described herein), they will also generally be referred toherein as “CDR sequences” (i.e., as CDR1 sequences, CDR2 sequences andCDR3 sequences, respectively). It should however be noted that theinvention in its broadest sense is not limited to a specific structuralrole or function that these stretches of amino acid residues may have inan amino acid sequence of the invention, as long as these stretches ofamino acid residues allow the amino acid sequence of the invention tobind to DR5. Thus, generally, the invention in its broadest sensecomprises any amino acid sequence that is capable of binding to DR5 andthat comprises one or more CDR sequences as described herein, and inparticular a suitable combination of two or more such CDR sequences,that are suitably linked to each other via one or more further aminoacid sequences, such that the entire amino acid sequence forms a bindingdomain and/or binding unit that is capable of binding to DR5. It shouldhowever also be noted that the presence of only one such CDR sequence inan amino acid sequence of the invention may by itself already besufficient to provide an amino acid sequence of the invention that iscapable of binding to DR5; reference is, e.g., again made to theso-called “Expedite fragments” described in WO 03/050531.

In one embodiment, the amino acid sequence of the invention may be anamino acid sequence that comprises at least one amino acid sequence thatis chosen from the group consisting of the CDR1 sequences, CDR2sequences and CDR3 sequences that are described herein (or any suitablecombination thereof). In particular, an amino acid sequence of theinvention may be an amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast one amino acid sequence that is chosen from the group consistingof the CDR1 sequences, CDR2 sequences and CDR3 sequences that aredescribed herein (or any suitable combination thereof).

In one embodiment, the amino acid sequence of the invention may be anyamino acid sequence that comprises at least one stretch of amino acidresidues, in which said stretch of amino acid residues has an amino acidsequence that corresponds to the sequence of at least one of the CDRsequences described herein. Such an amino acid sequence may or may notcomprise an immunoglobulin fold. For example, and without limitation,such an amino acid sequence may be a suitable fragment of animmunoglobulin sequence that comprises at least one such CDR sequence,but that is not large enough to form a (complete) immunoglobulin fold(reference is, e.g., again made to the “Expedite fragments” described inWO 03/050531). Alternatively, such an amino acid sequence may be asuitable “protein scaffold” that comprises least one stretch of aminoacid residues that corresponds to such a CDR sequence (i.e., as part ofits antigen binding site). Suitable scaffolds for presenting amino acidsequences will be clear to the skilled person, and, e.g., comprise,without limitation, to binding scaffolds based on or derived fromimmunoglobulins (i.e., other than the immunoglobulin sequences alreadydescribed herein), protein scaffolds derived from protein A domains(e.g., such as AFFIBODIES™) tendamistat, fibronectin, includingfibronectin type III domain, lipocalin, CTLA-4, T-cell receptors,designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat.Biotech 2005, Vol. 23: 1257), and binding moieties based on DNA or RNAincluding but not limited to DNA or RNA aptamers (Ulrich et al., CombChem. High Throughput Screen 2006 9(8): 619-32).

In one embodiment, any amino acid sequence herein that comprises one ormore of these CDR sequences can specifically bind to DR5. In oneembodiment it can bind to DR5 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value) as provided herein.

In one embodiment, an inventive composition herein may be any amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least two amino acid sequences thatare chosen from the group consisting of the CDR1 sequences describedherein, the CDR2 sequences described herein and the CDR3 sequencesdescribed herein, such that (1) when the first amino acid sequence ischosen from the CDR1 sequences described herein, the second amino acidsequence is chosen from the CDR2 sequences described herein or the CDR3sequences described herein; (2) when the first amino acid sequence ischosen from the CDR2 sequences described herein, the second amino acidsequence is chosen from the CDR1 sequences described herein or the CDR3sequences described herein; or (3) when the first amino acid sequence ischosen from the CDR3 sequences described herein, the second amino acidsequence is chosen from the CDR1 sequences described herein or the CDR3sequences described herein.

In one embodiment, an inventive composition herein may be amino acidsequences that comprise at least one antigen binding site, wherein saidantigen binding site comprises at least three amino acid sequences thatare chosen from the group consisting of the CDR1 sequences describedherein, the CDR2 sequences described herein and the CDR3 sequencesdescribed herein, such that the first amino acid sequence is chosen fromthe CDR1 sequences described herein, the second amino acid sequence ischosen from the CDR2 sequences described herein, and the third aminoacid sequence is chosen from the CDR3 sequences described herein.Preferred combinations of CDR1, CDR2 and CDR3 sequences will becomeclear from the further description herein. As will be clear to theskilled person, such an amino acid sequence is preferably animmunoglobulin sequence (as further described herein), but it may, e.g.,also be any other amino acid sequence that comprises a suitable scaffoldfor presenting said CDR sequences.

In one embodiment, an inventive composition herein relates to an aminoacid sequence directed against DR5, and in particular against DR5 asprovided in public databases such as the e.g. NCBI protein databaseaccession number BAA33723, wherein the amino acid sequence comprises oneor more stretches of amino acid residues chosen from the groupconsisting of:

(a) the amino acid sequences of SEQ ID NO's: 41 to 44;

(b) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 41 to 44;

(c) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 41 to 44;

(d) amino acid sequences of SEQ ID NO's: 51 to 55;

(e) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 51 to 55;

(f) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 51 to 55;

(g) the amino acid sequences of SEQ ID NO's: 63 to 68;

(h) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 63 to 68;

(i) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 63 to 68;

or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

(1) any amino acid substitution in such an amino acid sequence accordingto b) and/or c) is preferably, and compared to the corresponding aminoacid sequence according to a), a conservative amino acid substitution;and/or

(2) the amino acid sequence according to b) and/or c) preferably onlycontains amino acid substitutions, and no amino acid deletions orinsertions, compared to the corresponding amino acid sequence accordingto a); and/or

(3) the amino acid sequence according to b) and/or c) may be an aminoacid sequence that is derived from an amino acid sequence according toa) by means of affinity maturation using one or more techniques ofaffinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

(1) any amino acid substitution in such an amino acid sequence accordingto e) and/or f) is preferably, and compared to the corresponding aminoacid sequence according to d), a conservative amino acid substitution;and/or

(2) the amino acid sequence according to e) and/or f) preferably onlycontains amino acid substitutions, and no amino acid deletions orinsertions, compared to the corresponding amino acid sequence accordingto d); and/or

(3) the amino acid sequence according to e) and/or f) may be an aminoacid sequence that is derived from an amino acid sequence according tod) by means of affinity maturation using one or more techniques ofaffinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

(1) any amino acid substitution in such an amino acid sequence accordingto h) and/or i) is preferably, and compared to the corresponding aminoacid sequence according to g), a conservative amino acid substitution;and/or

(2) the amino acid sequence according to h) and/or i) preferably onlycontains amino acid substitutions, and no amino acid deletions orinsertions, compared to the corresponding amino acid sequence accordingto g); and/or

(3) the amino acid sequence according to h) and/or i) may be an aminoacid sequence that is derived from an amino acid sequence according tog) by means of affinity maturation using one or more techniques ofaffinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to a), b), c), d),e), f), g), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

(i) the amino acid sequences of SEQ ID NO's: 41 to 44;

(ii) the amino acid sequences of SEQ ID NO's: 51 to 55; and

(iii) the amino acid sequences of SEQ ID NO's: 63 to 68;

or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against DR5.

In one non-limiting aspect, the invention relates to an amino acidsequence directed against DR5, that comprises two or more stretches ofamino acid residues chosen from the group consisting of:

a) the amino acid sequences of SEQ ID NO's: 41 to 44;

b) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 41 to 44;

c) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 41 to 44;

d) the amino acid sequences of SEQ ID NO's: 51 to 55;

e) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 51 to 55;

f) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 51 to 55;

g) the amino acid sequences of SEQ ID NO's: 63 to 68;

h) amino acid sequences that have at least 90% amino acid identity withat least one of the amino acid sequences of SEQ ID NO's: 63 to 68;

i) amino acid sequences that have 3, 2, or 1 amino acid difference withat least one of the amino acid sequences of SEQ ID NO's: 63 to 68;

such that (1) when the first stretch of amino acid residues correspondsto one of the amino acid sequences according to a), b) or c), the secondstretch of amino acid residues corresponds to one of the amino acidsequences according to d), e), f), g), h) or i); (2) when the firststretch of amino acid residues corresponds to one of the amino acidsequences according to d), e) or f), the second stretch of amino acidresidues corresponds to one of the amino acid sequences according to a),b), c), g), h) or i); or (3) when the first stretch of amino acidresidues corresponds to one of the amino acid sequences according to g),h) or i), the second stretch of amino acid residues corresponds to oneof the amino acid sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

(i) the amino acid sequences of SEQ ID NO's: 41 to 44;

(ii) the amino acid sequences of SEQ ID NO's: 51 to 55; and

(iii) the amino acid sequences of SEQ ID NO's: 63 to 68;

such that, (1) when the first stretch of amino acid residues correspondsto one of the amino acid sequences of SEQ ID NO's: 41 to 44, the secondstretch of amino acid residues corresponds to one of the amino acidsequences of SEQ ID NO's: 51 to 55 or of SEQ ID NO's: 63 to 68; (2) whenthe first stretch of amino acid residues corresponds to one of the aminoacid sequences of SEQ ID NO's: 51 to 55, the second stretch of aminoacid residues corresponds to one of the amino acid sequences of SEQ IDNO's: 41 to 44 or of SEQ ID NO's: 63 to 68; or (3) when the firststretch of amino acid residues corresponds to one of the amino acidsequences of SEQ ID NO's: 63 to 68, the second stretch of amino acidresidues corresponds to one of the amino acid sequences of SEQ ID NO's:41 to 44 or of SEQ ID NO's: 51 to 55.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against DR5.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising two ormore amino acid sequences according to a), b), c), d), e), f), g), h) ori), respectively. In one embodiment, the amino acid sequence is adimeric variant of a NB agent. In one embodiment, the monomers thatcomprise the dimer are operably linked in a single reading frame. In oneembodiment, the linker sequence joining the monomers is a multimer ofthe sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82), or acombination of both, or is selected from a linker sequences providedherein as SEQ ID NOs: 44, 45 or 46, or as known in the art.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising three ormore amino acid sequences according to a), b), c), d), e), f), g), h) ori), respectively. In one embodiment, the amino acid sequence is atrimeric variant of a NB agent. In one embodiment, the monomers thatcomprise the trimer are operably linked in a single reading frame. Inone embodiment, the linker sequence joining the monomers is a multimerof the sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82), or acombination of both, or is selected from a linker sequences providedherein as SEQ ID NOs: 44, 45 or 46, or as known in the art.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising four ormore amino acid sequences according to a), b), c), d), e), f), g), h) ori), respectively. In one embodiment, the amino acid sequence is atetrameric variant of a NB agent. In one embodiment, the monomers thatcomprise the tetramer are operably linked in a single reading frame. Inone embodiment, the linker sequence joining the monomers is a multimerof the sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82), or acombination of both, or is selected from a linker sequences providedherein as SEQ ID NOs: 44, 45 or 46, or as known in the art.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising five ormore amino acid sequences according to a), b), c), d), e), f), g), h) ori), respectively. In one embodiment, the amino acid sequence is apentameric variant of a NB agent. In one embodiment, the monomers thatcomprise the pentamer are operably linked in a single reading frame. Inone embodiment, the linker sequence joining the monomers is a multimerof the sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82), or acombination of both, or is selected from a linker sequences providedherein as SEQ ID NOs: 44, 45 or 46, or as known in the art.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising six ormore amino acid sequences according to a), b), c), d), e), f), g), h) ori), respectively. In one embodiment, the amino acid sequence is ahexameric variant of a NB agent. In one embodiment, the monomers thatcomprise the hexamer are operably linked in a single reading frame. Inone embodiment, the linker sequence joining the monomers is a multimerof the sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82), or acombination of both, or is selected from a linker sequences providedherein as SEQ ID NOs: 44, 45 or 46, or as known in the art.

In one embodiment, in particular as it relates to the precedingparagraphs, the invention related to an amino acid comprising two,three, four, five six, seven, eight, nine, ten or more amino acidsequences according to a), b), c), d), e), f), g), h) or i),respectively. In one embodiment, the amino acid sequence is a multimericvariant of a NB agent. In one embodiment, monomers that comprise themultimer are operably linked in a single reading frame. In oneembodiment, the linker sequence joining the monomers is selected from atleast one of the sequence GGGGS (SEQ ID NO: 81) or GGGS (SEQ ID NO: 82),or a combination of both, or is selected from a linker sequencesprovided herein as SEQ ID NOs: 44, 45 or 46, or as known to one skilledin the art.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 90% amino acid identity,more preferably at least 90% amino acid identity, such as at least 95%amino acid identity or more or even essentially 100% amino acid identitywith the CDR sequences of at least one of the amino acid sequences ofSEQ ID NO's: 1-22, 26-40, 87-88, and 102-103. This degree of amino acididentity can, e.g., be determined by determining the degree of aminoacid identity (in a manner described herein) between said amino acidsequence and one or more of the sequences of SEQ ID NO's: 1-22, 26-40,87-88, and 102-103, in which the amino acid residues that form theframework regions are disregarded. Also, such amino acid sequences canbe humanized or modified as further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind to DR5; and more in particular bind to DR5 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as provided herein.

In one non-limiting aspect, the invention relates to an amino acidsequence that essentially consists of four framework regions (FR1 toFR4, respectively) and three complementarity determining regions (CDR1to CDR3, respectively), in which the CDR sequences of said amino acidsequence have at least 70% amino acid identity, preferably at least 90%amino acid identity, more preferably at least 90% amino acid identity,such as at least 95% amino acid identity or more, at least 98% aminoacid identity or more, at least 99% amino acid identity or more, or evenessentially 100% amino acid identity with the CDR sequences of at leastone of the amino acid sequences of SEQ ID NO's: 1-22, 26-40, 87-88, and102-103. This degree of amino acid identity can, e.g., be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 1-22, 26-40, 87-88, and 102-103, in which theamino acid residues that form the framework regions are disregarded.Such amino acid sequences of the invention can be as further modified asdescribed herein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, e.g., on thebasis the standard handbooks and the further disclosure and artreferences mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional VH sequences thathave been camelized.

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a NB agent (including but not limited to V_(H)and/or V_(HH) sequence). Again, suitable framework sequences will beclear to the skilled person, e.g., on the basis the standard handbooksand the further disclosure and art references mentioned herein.

In one embodiment, as generally described herein for the amino acidsequences of the invention, it is possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one aspect, such a fragment comprises a single CDR sequence asdescribed herein (and in particular a CDR3 sequence), that is flanked oneach side by (part of) a framework sequence (and in particular, part ofthe framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions). In one embodiment, such aconstruct is an Expedite fragment. For a further description of these“Expedite fragments”, reference is again made to WO 03/050531, as wellas to the US provisional application of Ablynx N.V. entitled “Peptidescapable of binding to serum proteins” of Ablynx N.V. (inventors: Revetset al.) filed on Dec. 5, 2006.

In one aspect, the invention relates to a NB agent, and in particular aprotein or polypeptide that comprises or essentially consists of one ormore amino acid sequences of the invention (or suitable fragmentsthereof), and optionally further comprises one or more other groups,residues, moieties or binding units. As will become clear to the skilledperson from the further disclosure herein, such further groups,residues, moieties, binding units or amino acid sequences may or may notprovide further functionality to the amino acid sequence of theinvention (and/or to the compound or construct in which it is present)and may or may not modify the properties of the amino acid sequence ofthe invention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences. Evenmore preferably, said one or more other groups, residues, moieties orbinding units are chosen from the group consisting of domain antibodies,amino acid sequences that are suitable for use as a domain antibody,single domain antibodies, amino acid sequences that are suitable for useas a single domain antibody, “dAb's”, amino acid sequences that aresuitable for use as a dAb, or camelid V_(HH) constructs.

In one embodiment, such groups, residues, moieties or binding units maye.g., be chemical groups, residues, moieties, which may or may not bythemselves be biologically and/or pharmacologically active. For example,and without limitation, such groups may be linked to the one or moreamino acid sequences of the invention so as to provide a “derivative” ofan amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion protein or fusionpolypeptide. The amino acid sequences of such linkers may be as providedherein, or may be one known to a person skilled in the art.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, e.g.,on the basis of the methods and techniques further described herein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

According to one preferred aspect, a compound of the invention is amultivalent polypeptide that contains two or more (preferably three ormore, four or more, five or more, six or more, eight or more, and/or tenor more) amino acid sequences directed against the DR5 (e.g. amino acidsequences as provided herein), optionally linked via one or moresuitable linkers (which may be as further described herein) or via across-linking technique or covalent bond created in a manner known toone skilled in the art. In one or more alternative embodiments, themultivalent polypeptide contains an optional subunit that binds to atarget other than DR5.

In particular, a compound of the invention may be such a multivalentpolypeptide which is capable of inducing, triggering, increasing orenhancing the signaling mediated by the DR5, and more in particular oftriggering or inducing apoptosis in the cell on which the DR5 ispresent. In one embodiment, the multivalent polypeptide consists ofthree monomeric DR5 binding units that are operably linked, therebycreating a trimeric polypeptide. In one embodiment, the multivalentpolypeptide consists of four monomeric DR5 binding units that areoperably linked, thereby creating a tetrameric polypeptide. In oneembodiment, the multivalent polypeptide consists of five monomeric DR5binding units that are operably linked, thereby creating a pentamericpolypeptide. In one embodiment, the multivalent polypeptide consists ofsix monomeric DR5 binding units that are operably linked, therebycreating a hexameric polypeptide. In one embodiment, the multivalentpolypeptide consists of seven, eight, nine, ten or more monomeric DR5binding units that are operably linked, thereby creating additionalvariants of a multimeric protein.

As already mentioned herein, such multimeric polypeptide may be furthersuch that it can bind to the binding site of TRAIL on the DR5, and/orsuch that they can compete with TRAIL for binding to the DR5.

The multivalent polypeptide may also be such that it does not bind tothe binding site for TRAIL on the DR5, and/or essentially does notprevent or inhibit the binding of TRAIL to the DR5, and/or does notcompete with TRAIL for binding to the DR5, and/or even be such that theyincrease or enhance the signaling that is mediated by TRAIL and the DR5upon binding of TRAIL to its receptor (which may again result in asynergistic effect upon the signaling mediated by the DR5). In oneembodiment, said multivalent polypeptide binds to DR5 with a higheraffinity than a monovalent composition.

In the multivalent polypeptides of the invention, the amino acidsequences of the invention that form the binding units may each bedirected against the same epitope on the DR5 receptor, e.g., an epitopeof DR5, or against different epitopes on the DR5.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somenon-limiting examples of such compounds and polypeptides will becomeclear to the skilled person based on the further disclosure herein, ande.g., comprise amino acid sequences or polypeptides of the inventionthat have been chemically modified to increase the half-life thereof(for example, by means of pegylation); amino acid sequences of theinvention that comprise at least one additional binding site for bindingto a serum protein (such as serum albumin); or polypeptides of theinvention that comprise at least one amino acid sequence of theinvention that is linked to at least one moiety (and in particular atleast one amino acid sequence) that increases the half-life of the aminoacid sequence of the invention. Examples of polypeptides of theinvention that comprise such half-life extending moieties or amino acidsequences will become clear to the skilled person based on the furtherdisclosure herein; and, e.g., include, without limitation, polypeptidesin which the one or more amino acid sequences of the invention aresuitable linked to one or more serum proteins or fragments thereof (suchas (human) serum albumin or suitable fragments thereof) or to one ormore binding units that can bind to serum proteins (such as, e.g.,domain antibodies, amino acid sequences that are suitable for use as adomain antibody, single domain antibodies, amino acid sequences that aresuitable for use as a single domain antibody, (dAb's), amino acidsequences that are suitable for use as a dAb, or NANOBODIES™ that canbind to serum proteins such as serum albumin (such as human serumalbumin), serum immunoglobulins such as IgG, or transferrine; referenceis made to the further description and references mentioned herein);polypeptides in which an amino acid sequence of the invention is linkedto an Fc portion (such as a human Fc) or a suitable part or fragmentthereof; or polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more small proteins orpeptides that can bind to serum proteins (such as, without limitation,the proteins and peptides described in WO 91/01743, WO 01/45746, WO02/076489 and to the US provisional application of Ablynx N.V. entitled“Peptides capable of binding to serum proteins” of Ablynx N.V. filed onDec. 5, 2006.

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, e.g., at least 10times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and may,e.g., be in the form of a genetic construct, e.g., a CDR fragment or ina vector, as further described herein. In one embodiment, the nucleicacid sequences of the invention encode the NB constructs of any one ormore polypeptide disclosed in any one or more of Tables 1 through 4. Inone embodiment, the nucleic acid sequence of the invention encodes anyone or more construct provided in SEQ ID NOs: 1-72, 81-82 and 87-92. Inone embodiment, the nucleic acid sequences of the invention are codonoptimized for expression in the host cell or host organism of interest.In one embodiment, the codon optimization is for expression in amammalian cell. In one embodiment, the codon optimization is forexpression in a yeast cell. In one embodiment, the codon optimization isfor expression in E. coli.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof), atleast one compound of the invention and/or at least one nucleic acid ofthe invention, and optionally one or more further components of suchcompositions known per se, i.e., depending on the intended use of thecomposition. Such a product or composition may, e.g., be apharmaceutical composition (as described herein), a veterinarycomposition or a product or composition for diagnostic use (as alsodescribed herein). Some non-limiting examples of such products orcompositions are provided herein.

In one embodiment the invention relates to the use of a NB construct, orof a composition comprising the same, in methods or compositions formodulating DR5, either in vitro (e.g. in an in vitro or cellular assay)or in vivo (e.g. in an a single cell or in a multicellular organism, andin particular in a mammal, and more in particular in a human being, suchas in a human being that is at risk of or suffers from a diseases anddisorders associated with DR5).

The invention also relates to methods for modulating DR5, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a diseases and disorders associated withDR5), which method comprises at least the step of contacting DR5 with atleast one amino acid sequence, NB construct of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate DR5, with at least one amino acid sequence, NB construct ofthe invention.

The invention also relates to the use of an one amino acid sequence, NBconstruct of the invention in the preparation of a composition (such as,without limitation, a pharmaceutical composition or preparation asfurther described herein) for modulating DR5, either in vitro (e.g. inan in vitro or cellular assay) or in vivo (e.g. in an a single cell ormulticellular organism, and in particular in a mammal, and more inparticular in a human being, such as in a human being that is at risk ofor suffers from a diseases and disorders associated with DR5).

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or completely inhibiting the activityof, or alternatively increasing the activity of, DR5, as measured usinga suitable in vitro, cellular or in vivo assay (such as those mentionedherein). In particular, “modulating” or “to modulate” may mean eitherreducing or completely inhibiting the activity of, or alternativelyincreasing the activity of DR5, as measured using a suitable in vitro,cellular or in vivo assay (such as those mentioned herein), by at least1%, preferably at least 5%, such as at least 10% or at least 25%, e.g.,by at least 50%, at least 60%, at least 70%, at least 80%, or at least90% or more, compared to activity of DR5 in the same assay under thesame conditions but without the presence of the NB construct of theinvention. In a main embodiment, DR5 activity, especially includingDR5-mediated signaling, requires the formation of a trimer of DR5 on thecell surface. In one embodiment, a NB construct herein acts to modulateDR5 activity by promoting the formation of a DR5 homotrimer and therebyincreasing the DR5-mediated initiation of apoptosis, e.g., via theextrinsic cell death pathway.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of DR5 for one or moreof its targets, ligands or substrates; and/or effecting a change (whichmay either be an increase or a decrease) in the sensitivity of DR5 forone or more conditions in the medium or surroundings in which DR5 ispresent (such as pH, ion strength, the presence of co-factors, etc.),compared to the same conditions but without the presence of the aminoacid sequence, NB construct of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, such as the assaysdescribed herein or in the art references cited herein.

“Modulating” may also mean effecting a change (i.e., an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which DR5 (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signaling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, such as the assays described herein orin the art references cited herein. In particular, an action as anagonist or antagonist may be such that an intended biological orphysiological activity is increased or decreased, respectively, by atleast 1%, preferably at least 5%, such as at least 10% or at least 25%,e.g., by at least 50%, at least 60%, at least 70%, at least 80%, or atleast 90% or more, compared to the biological or physiological activityin the same assay under the same conditions but without the presence ofthe NB construct of the invention.

Modulating may, e.g., involve reducing or inhibiting the binding of DR5to one of its substrates or ligands and/or competing with a naturalligand, substrate for binding to DR5. Modulating may also involveactivating DR5 or the mechanism or pathway in which it is involved.Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

a) providing a set, collection or library of amino acid sequences;

b) screening said set, collection or library of amino acid sequences foramino acid sequences that can bind to and/or have affinity for DR5; and

c) isolating the amino acid sequence(s) that bind to and/or haveaffinity for DR5.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, e.g., derived from a mammal that has beensuitably immunized with DR5 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, e.g., on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23(9): 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

a) providing a collection or sample of cells expressing amino acidsequences;

b) screening said collection or sample of cells for cells that expressan amino acid sequence that can bind to and/or have affinity for DR5;and

c) either (1) isolating said amino acid sequence; or (2) isolating fromsaid cell a nucleic acid sequence that encodes said amino acid sequence,followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may, e.g., be a collectionor sample of B-cells. Also, in this method, the sample of cells may bederived from a mammal that has been suitably immunized with DR5 or witha suitable antigenic determinant based thereon or derived therefrom,such as an antigenic part, fragment, region, domain, loop or otherepitope thereof. In one particular aspect, said antigenic determinantmay be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is, e.g., made to EP 0 542 810,WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) ispreferably performed using a flow cytometry technique such as FACS. Forthis, reference is, e.g., made to Lieby et al., Blood, 97(12), 3820(2001).

In another aspect, the method for generating an amino acid sequencedirected against DR5 may comprise at least the steps of:

a) providing a set, collection or library of nucleic acid sequencesencoding amino acid sequences;

b) screening said set, collection or library of nucleic acid sequencesfor nucleic acid sequences that encode an amino acid sequence that canbind to and/or has affinity for DR5; and

c) isolating said nucleic acid sequence, followed by expressing saidamino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may, e.g., be a set, collectionor library of nucleic acid sequences encoding a naïve set, collection orlibrary of immunoglobulin sequences; a set, collection or library ofnucleic acid sequences encoding a synthetic or semi-synthetic set,collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. In one example, the set, collection or libraryof nucleic acid sequences encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In one aspect of this method, the set, collection or library of aminoacid sequences may be an immune set, collection or library of nucleicacid sequences, e.g., derived from a mammal that has been suitablyimmunized with DR5 or with a suitable antigenic determinant basedthereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may, e.g.,encode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, e.g., on the basis of the further disclosure herein. Reference isalso made to the review by Hoogenboom in Nature Biotechnology, 23(9):1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises one ofthe above methods and in addition at least the steps of determining thenucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina known manner, such as by expression in a suitable host cell or hostorganism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with DR5. Some preferred but non-limiting applications anduses will become clear from the further description herein.

In one embodiment, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder.

In one embodiment, the invention relates to one or more of the aminoacid sequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein for use in therapy ofa subject at risk for or suffering from a disease or disorder that canbe prevented or treated by administering, to the subject in needthereof, a pharmaceutically effective amount of an amino acid sequence,compound, construct or polypeptide as described herein as an NB agent orNB construct.

In one embodiment, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of diseases anddisorders associated with DR5.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein, in which theinvention is described and discussed in more detail.

In certain embodiments, inventive V_(HH) variants of the NB agentsgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the inventive compositions of theinvention. However, it will be clear to the skilled person that the moregeneral aspects of the teaching below can also be applied (eitherdirectly or analogously) to other amino acid sequences of the invention.

DEFINITIONS

In the present description, examples and claims, the followingdefinitions are generally applied, unless indicated otherwise. As usedbelow and throughout the text, the use of a term or phrase in thesingular is meant to incorporate the meaning of the plural, and viceversa, unless noted otherwise or apparent through context.

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis made, e.g., to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10 Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein. Additional references include,e.g., the following reviews: Presta, Adv. Drug Deliv. Rev. 2006, 58(5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irvinget al., J. Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al.,Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol.,2005, 26(1), 31-43, which describe techniques for protein engineering,such as affinity maturation and other techniques for improving thespecificity and other desired properties of proteins such asimmunoglobulins.

Unless indicated otherwise, it is believed that all methods, steps,techniques and manipulations that are not specifically described indetail can be performed and have been performed in a manner known perse, and will be clear to the skilled person. Reference is again made,e.g., to the standard handbooks and the general background art mentionedherein and to the further references cited therein. Amino acid residueswill be indicated according to the standard three-letter or one-letteramino acid code as provided by the IUPAC Code Tables.

Unless indicated otherwise, the term “immunoglobulin sequence”, whetherused herein to refer to a heavy chain antibody or to a conventionalfour-chain antibody, is used as a general term to include both thefull-size antibody, the individual chains thereof, as well as all parts,domains or fragments thereof (including but not limited toantigen-binding domains or fragments such as V_(HH) domains orV_(H)/V_(L) domains, respectively).

The term “sequence” as used herein (for example in terms like“immunoglobulin sequence”, “antibody sequence”, “variable domainsequence”, “sdAb sequence”, “V_(H) sequence”, “V_(HH) sequence” or“protein sequence”), is generally used to include both the relevantamino acid sequence as well as nucleic acid sequences or nucleotidesequences encoding the same, unless the context requires a more limitedinterpretation.

Unless indicated otherwise, the terms “nucleotide sequence” and “nucleicacid” generally is used interchangeably to refer to a polymer ofdeoxyribonucleic acids (DNA) or ribonucleic acids (RNA), unless thecontext requires a more limited interpretation.

For the purposes of comparing two or more nucleotide sequences, thepercentage of “sequence identity” between a first nucleotide sequenceand a second nucleotide sequence may be calculated or determined, e.g.,by taking the number of nucleotides in the first nucleotide sequencethat are identical to the nucleotides at the corresponding positions inthe second nucleotide sequence, dividing that number by the total numberof nucleotides in the first nucleotide sequence and then multiplying by100%. Each deletion, insertion, substitution or addition of a nucleotidein the second nucleotide sequence—when compared to the first nucleotidesequence—is considered as a difference at a single nucleotide position.One skilled in the art may also use a suitable computer algorithm ortechnique such as, e.g., NCBI Blast v2.0, using standard settings.

With regard to framework 1 (FR1), it should be clear to the skilledperson that, for determining the degree of amino acid identity, theamino acid residues on positions 1 to 4 and 27 to 30 are preferablydisregarded.

In one embodiment, in determining the degree of sequence identitybetween two amino acid sequences, the skilled person may take intoaccount so-called “conservative” amino acid substitutions. Conservativesubstitutions are known in that art and are substitutions in which oneamino acid is substituted by another amino acid residue within the samegroup that have a common property. Conserved amino acid groups and theircommon properties are as follows: (a) small aliphatic, nonpolar orslightly polar residues, including Ala, Ser, Thr, Pro and Gly; (b)polar, negatively charged residues and their (uncharged) amides,including Asp, Asn, Glu and Gln; (c) polar, positively charged residues,including His, Arg and Lys; (d) large aliphatic, nonpolar residues,including Met, Leu, He, Val and Cys; and (e) aromatic residues,including Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp intoGlu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Natl. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157:105-132, 198 1, and Goldman et al., Ann. Rev. Biophys. Chem. 15:321-353, 1986. Information on the primary, secondary and tertiarystructure of camelid V_(HH) constructs is given in the generalbackground art cited above, and the crystal structure of such a V_(HH)construct from a llama is, e.g., given by Desmyter et al., NatureStructural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., NaturalStructural Biology (1996); 3, 752-757; and Decanniere et al., Structure,Vol. 7, 4, 361 (1999). Further information about some of the amino acidresidues that in conventional V_(H) domains form the V_(H)/V_(L)interface and potential camelizing substitutions on these positions canbe found in the art cited above.

Amino acid sequences and nucleic acid sequences are said to be “exactlythe same” if they have 100% sequence identity over their entire length.

When comparing two amino acid sequences, the term “amino aciddifference” refers to an insertion, deletion or substitution of a singleamino acid residue on a position of the first sequence, compared to thesecond sequence; it being understood that two amino acid sequences cancontain one, two or more such amino acid differences;

When a nucleotide sequence or amino acid sequence is said to “comprise”another nucleotide sequence or amino acid sequence, respectively, or to“essentially consist of another nucleotide sequence or amino acidsequence, this generally means that the first mentioned nucleotidesequence or amino acid sequence has within its sequence a stretch ofnucleotides or amino acid residues, respectively, that has the samenucleotide sequence or amino acid sequence, respectively, as the lattersequence, irrespective of how the first mentioned sequence has actuallybeen generated or obtained (which may, e.g., be by any suitable methoddescribed herein).

The term “in essentially isolated form” for a nucleic acid or amino acidsequence is separated from at least one other component with which it isusually associated in a source or medium, compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained. The “at least one other component” may beanother nucleic acid, another protein/polypeptide, another biologicalcomponent or macromolecule or at least one contaminant, impurity orminor component. In one embodiment, a nucleic acid sequence or aminoacid sequence is considered “essentially isolated” when it has beenpurified at least 2-fold, in particular at least 10-fold, more inparticular at least 100-fold, and up to 1000-fold or more. A nucleicacid sequence or amino acid sequence that is “in essentially isolatedform” is preferably essentially homogeneous, as determined using asuitable technique, such as a suitable chromatographical technique, suchas polyacrylamide-gel electrophoresis.

The terms “domain” and “binding domain” as used herein generally refersto a globular region of an antibody chain, and in particular to aglobular region of a heavy chain antibody, or to a polypeptide thatessentially consists of such a globular region. Usually, such a domainwill comprise peptide loops (for example 3 or 4 peptide loops)stabilized, e.g., as a sheet or by disulfide bonds.

The terms “antigenic determinant” and “epitope”, which may also be usedinterchangeably herein, refers to the epitope on the antigen recognizedby the antigen-binding molecule (such as a NB construct of theinvention) and more in particular by the antigen-binding site of saidmolecule.

An amino acid sequence (such as a NB construct of the invention, aV_(HH) or V_(H) construct, an antibody, or generally an antigen bindingprotein or polypeptide or a fragment thereof) that can (specifically)bind to, that has affinity for, and/or that has specificity for aparticular antigenic determinant, epitope, antigen or protein (or for atleast one part, fragment or epitope thereof) is said to be “against” or“directed against” said antigenic determinant, epitope, antigen orprotein.

The term “specificity” as mentioned therein refers to the number ofdifferent types of antigens or antigenic determinants to which aparticular antigen-binding molecule or antigen-binding protein (such asa NB construct of the invention) molecule can bind. The specificity ofan antigen-binding protein can be determined based on affinity and/oravidity. Typically, antigen-binding proteins (such as the NB constructof the invention) will bind to their antigen with a dissociationconstant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²moles/liter (i.e., with an association constant (K_(A)) of 10⁵ to 10¹²liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more andmore preferably 10⁸ to 10¹² liter/moles). Any K_(D) value greater than10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹) liters/mol isgenerally considered to indicate non-specific binding. Preferably, a NBconstruct of the invention will bind to its desired antigen with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. “Specific binding” of anantigen-binding protein to an antigen or antigenic determinant can bedetermined in any suitable manner known per se, including, e.g.,Scatchard analysis and/or competitive binding assays, such asradioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwichcompetition assays, and the different variants thereof known per se inthe art; as well as the other techniques mentioned herein.

“Affinity”, represented by the equilibrium constant for the dissociationof an antigen with an antigen-binding protein (K_(D)), is a measure forthe binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)). As will be clear to the skilled person, affinity canbe determined in a manner known per se, depending on the specificantigen of interest.

“Avidity” is the measure of the strength of binding between anantigen-binding molecule (such as a NB construct of the invention) andthe pertinent antigen. Avidity is related to both the affinity betweenan antigenic determinant and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule.

The dissociation constant may be the actual or apparent dissociationconstant. Methods for determining the dissociation constant are known inthe art.

The phrase “specifically binds” or “selectively binds,” when used in thecontext of describing the interaction between an antigen (e.g., aprotein) and an NB construct, or functional fragment thereof, e.g.,having the disclosed CDRs in the framework of an antibody, antibodyfragment, or antibody-derived binding agent, refers to a bindingreaction that is determinative of the presence of the antigen in aheterogeneous population of proteins and other biologics, e.g., in abiological sample, e.g., a blood, serum, plasma or tissue sample. Thus,under certain designated immunoassay conditions, the NB construct,antibodies or binding agents with a particular binding specificity bindto a particular antigen at least two times the background and do notsubstantially bind in a significant amount to other antigens present inthe sample. In one embodiment, under designated immunoassay conditions,the NB construct, antibody or binding agents with a particular bindingspecificity bind to a particular antigen at least ten (10) times thebackground and do not substantially bind in a significant amount toother antigens present in the sample. Specific binding to an NBconstruct, antibody or binding agent under such conditions may requirethe NB construct, antibody or binding agent to have been selected forits specificity for a particular protein. As desired or appropriate,this selection may be achieved by subtracting out NB constructs orantibodies that cross-react with, e.g., DR5 molecules from other species(e.g., mouse or rat) or other polypeptides that are Death Receptorfamily members or subtypes. Alternatively, in some embodiments, NBconstructs, antibodies or antibody fragments are selected thatcross-react with certain desired molecules.

A variety of immunoassay formats may be used to select NB constructsthat are specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Using Antibodies, A Laboratory Manual (1998), for a descriptionof immunoassay formats and conditions that can be used to determinespecific immunoreactivity) and can likewise be used for NB constructbinding analysis. Typically a specific or selective binding reactionwill produce a signal at least twice over the background signal and moretypically at least than 10 to 100 times over the background.

In addition to the affinity constant (K_(A)) described herein, anDR5-binding NB construct of the invention typically also has adissociation rate constant (K_(D)) (k_(off)/k_(on)) of less than5×10⁻²M, less than 10⁻²M, less than 5×10⁻³M, less than 10⁻³M, less than5×10⁻⁴M, less than 10⁻⁴M, less than 5×10⁻⁵M, less than 10⁻⁵M, less than5×10⁻⁶M, less than 10⁻⁶M, less than 5×10⁻⁷M, less than 10⁻⁷M, less than5×10⁻⁸M, less than 10⁻⁸M, less than 5×10⁻⁹M, less than 10⁻⁹M, less than5×10⁻¹ M less than 10⁻¹⁰M less than 5×10⁻¹¹M, less than 10⁻¹¹M, lessthan 5×10⁻¹²M, less than 10⁻¹²M, less than 5×10⁻¹³M, less than 10⁻¹³M,less than 5×10⁻¹⁴M, less than 10⁻¹⁴M, less than 5×10⁻¹⁵M, or less than10⁻¹⁵M or lower, and binds to DR5 with an affinity that is at leasttwo-fold greater than its affinity for binding to a non-specific antigen(e.g., human serum albumin “HSA”).

The half-life of an amino acid sequence, compound or polypeptide of theinvention generally refers to the time taken for the serum concentrationof the construct to be reduced by 50%, in vivo, e.g., due to degradationof the sequence or compound and/or clearance or sequestration of thesequence or compound by natural mechanisms. The in vivo half-life of anamino acid sequence, compound or polypeptide of the invention can bedetermined in any manner known per se, such as by pharmacokineticanalysis. Suitable techniques will be clear to the person skilled in theart. Half-life can be expressed using parameters such as thet_(1/2)-alpha, t_(1/2)-beta and the area under the curve (AUC). See theExperimental Part below, plus standard handbooks, such as Kenneth etal., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists,and Peters et al., Pharmacokinetic analysis: A Practical Approach(1996); Gibaldi & Perron, “Pharmacokinetics”, published by MarcelDekker, 2nd Rev. edition (1982). The terms “increase in half-life” or“increased half-life” refer to an increase in the t_(1/2)-beta, eitherwith or without an increase in the t_(1/2)-alpha and/or the AUC or both.

“Diseases and disorders associated with DR5” are as defined above andthroughout the specification, e.g., in paragraphs [0051]. While notbeing limited by theory, the mechanism of action of DR5 in such diseasesand/or disorders is believed to be as provided, e.g., in paragraph[0052]. Nonlimiting examples of contemplated diseases and disordersassociated with DR5 are as provided and throughout the specification,including e.g., in paragraph [0053], and others may become apparent toone skilled in the art based upon the teachings provided herein.

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, a target or antigen, asmeasured using a suitable in vitro, cellular or in vivo assay. Inparticular, “modulating” or “to modulate” may mean either reducing orinhibiting the activity of, or alternatively increasing a (relevant orintended) biological activity of, a target or antigen, as measured usinga suitable in vitro, cellular or in vivo assay (which will usuallydepend on the target or antigen involved), by at least 1%, preferably atleast 5%, such as at least 10% or at least 25%, e.g., by at least 50%,at least 60%, at least 70%, at least 80%, or at least 90% or more,compared to activity of the target or antigen in the same assay underthe same conditions but without the presence of the construct of theinvention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of a target or antigenfor one or more of its ligands, binding partners, partners forassociation into a homomultimeric or heteromultimeric form, orsubstrates; and/or effecting a change (which may either be an increaseor a decrease) in the sensitivity of the target or antigen for one ormore conditions in the medium or surroundings in which the target orantigen is present (such as pH, ion strength, the presence ofco-factors, etc.), compared to the same conditions but without thepresence of the construct of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, depending on the target orantigen involved.

In one embodiment, “modulating” may also mean effecting a change (i.e.,an activity as a neutralizing agent, as an agonist, as an antagonist oras a reverse agonist, respectively, depending on the target or antigenand the desired biological or physiological effect) with respect to oneor more biological or physiological mechanisms, effects, responses,functions, pathways or activities in which the target or antigen (or inwhich its substrate(s), ligand(s) or pathway(s) are involved, such asits signaling pathway or metabolic pathway and their associatedbiological or physiological effects) is involved. As will be clear tothe skilled person, such an action as a neutralizing agent, as anagonist or an antagonist may be determined in any suitable manner and/orusing any suitable (in vitro and usually cellular or in assay) assayknown per se, depending on the target or antigen involved. Inparticular, an action as an agonist or antagonist may be such that anintended biological or physiological activity is increased or decreased,respectively, by at least 1%, preferably at least 5%, such as at least10% or at least 25%, e.g., by at least 50%, at least 60%, at least 70%,at least 80%, or at least 90% or more, compared to the biological orphysiological activity in the same assay under the same conditions butwithout the presence of the construct of the invention.

Modulating may involve, e.g., allosteric modulation of the target orantigen; and/or reducing or inhibiting the binding of the target orantigen to one of its substrates or ligands and/or competing with anatural ligand, substrate for binding to the target or antigen.Modulating may also involve activating the target or antigen or themechanism or pathway in which it is involved. Modulating may alsoinvolve, e.g., effecting a change in respect of the folding orconfirmation of the target or antigen, or in respect of the ability ofthe target or antigen to fold, to change its confirmation (for example,upon binding of a ligand), to associate with other (sub)units, or todisassociate. Modulating may involve, e.g., effecting a change in theability of the target or antigen to transport other compounds or toserve as a channel for other compounds (such as ions).

Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

In respect of a target or antigen, the term “interaction site” on thetarget or antigen means a site, epitope, antigenic determinant, part,domain or stretch of amino acid residues on the target or antigen thatis a site for binding to a ligand, receptor or other binding partner, acatalytic site, a cleavage site, a site for allosteric interaction, asite involved in multimerization (such as homomerization orheteromerization) of the target or antigen; or any other site, epitope,antigenic determinant, part, domain or stretch of amino acid residues onthe target or antigen that is involved in a biological action ormechanism of the target or antigen. More generally, an “interactionsite” can be any site, epitope, antigenic determinant, part, domain orstretch of amino acid residues on the target or antigen to which anamino acid sequence or polypeptide of the invention can bind such thatthe target or antigen (and/or any pathway, interaction, signaling,biological mechanism or biological effect in which the target or antigenis involved) is modulated.

An amino acid sequence or polypeptide is said to be “specific for” afirst target or antigen compared to a second target or antigen when isbinds to the first antigen with an affinity (as described above, andsuitably expressed as a K_(D) value, K_(A) value, K_(off) rate and/orK_(on) rate) that is at least 10 times, such as at least 100 times, andpreferably at least 1000 times, and up to 10,000 times or more betterthan the affinity with which said amino acid sequence or polypeptidebinds to the second target or polypeptide. For example, the firstantigen may bind to the target or antigen with a K_(D) value that is atleast 10 times less, e.g., at least 100 times less, and preferably atleast 1000 times less, such as 10,000 times less or even a greater folddifference, than the K_(D) with which said amino acid sequence orpolypeptide binds to the second target or polypeptide. In oneembodiment, when an amino acid sequence or polypeptide is “specific for”a first target or antigen compared to a second target or antigen, it isdirected against said first target or antigen, but not directed againstsaid second target or antigen.

The terms “cross-block”, “cross-blocked” and “cross-blocking” are usedinterchangeably herein to mean the ability of an amino acid sequence orother binding agents (such as a polypeptide of the invention) tointerfere with the binding of other amino acid sequences or bindingagents of the invention to a given target. The extend to which an aminoacid sequence or other binding agents of the invention is able tointerfere with the binding of another to DR5, and therefore whether itcan be said to cross-block according to the invention, can be determinedusing competition binding assays. One particularly suitable quantitativeassay uses a Biacore machine which can measure the extent ofinteractions using surface plasmon resonance technology. Anothersuitable quantitative cross-blocking assay uses an ELISA-based approachto measure competition between amino acid sequence or another bindingagents in terms of their binding to the target.

A suitable assay for determining whether an amino acid sequence or otherbinding agent cross-blocks or is capable of cross-blocking according tothe invention is an exemplary Biacore assay or an exemplary ELISA assay.

An amino acid sequence is said to be “cross-reactive” for two differentantigens or antigenic determinants (such as serum albumin from twodifferent species of mammal, such as human serum albumin and cyno serumalbumin) if it is specific for both these different antigens orantigenic determinants.

By binding that is “essentially independent of the pH” is generallymeant herein that the association constant (K_(A)) of the amino acidsequence with respect to the serum protein (such as serum albumin) atthe pH value(s) that occur in a cell of an animal or human body (asfurther described herein) is at least 5%, such as at least 10%,preferably at least 25%, more preferably at least 50%, even morepreferably at least 60%, such as even more preferably at least 70%, suchas at least 80% or at least 90% or more (or even more than 100%, such asmore than 110%, more than 120% or even 130% or more, or even more than150%, or even more than 200%) of the association constant (K_(A)) of theamino acid sequence with respect to the same serum protein at the pHvalue(s) that occur outside said cell. Alternatively, by binding that is“essentially independent of the pH” is generally meant herein that thek_(off) rate (measured by Biacore—see e.g. Experiment 2) of the aminoacid sequence with respect to the serum protein (such as serum albumin)at the pH value(s) that occur in a cell of an animal or human body (ase.g. further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) isat least 5%, such as at least 10%, preferably at least 25%, morepreferably at least 50%, even more preferably at least 60%, such as evenmore preferably at least 70%, such as at least 80% or at least 90% ormore (or even more than 100%, such as more than 110%, more than 120% oreven 130% or more, or even more than 150%, or even more than 200%) ofthe k_(off) rate of the amino acid sequence with respect to the sameserum protein at the pH value(s) that occur outside said cell, e.g. pH7.2 to 7.4. By “the pH value(s) that occur in a cell of an animal orhuman body” is meant the pH value(s) that may occur inside a cell, andin particular inside a cell that is involved in the recycling of theserum protein. In particular, by “the pH value(s) that occur in a cellof an animal or human body” is meant the pH value(s) that may occurinside a (sub)cellular compartment or vesicle that is involved inrecycling of the serum protein (e.g. as a result of pinocytosis,endocytosis, transcytosis, exocytosis and phagocytosis or a similarmechanism of uptake or internalization into said cell), such as anendosome, lysosome or pinosome.

As further described herein, the total number of amino acid residues ina NB construct can be in the region of 110-120, is preferably 112-115,and is most preferably 113. It should however be noted that parts,fragments, analogs or derivatives (as further described herein) of a NBconstruct are not particularly limited as to their length and/or size,as long as such parts, fragments, analogs or derivatives meet thefurther requirements outlined herein, and are optionally suitable forthe purposes herein.

As mentioned herein “preferred” (or “more preferred”, “even morepreferred”, etc.) apply to specific NB compounds or variants thereof, ortheir uses, for a variety of embodiments described herein, or forparticular embodiments if so described.

The amino acid residues of a NB construct are numbered according to thegeneral numbering for V_(H) domains as given by Kabat et al. “Sequenceof proteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91, as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, J. Immunol.Methods 2000 Jun. 23; 240 (1-2): 185-195 (see, e.g., FIG. 2 of thispublication). Accordingly, in general a FR1 of a NB construct comprisesthe amino acid residues at positions 1-30, a CDR1 of a NB constructcomprises the amino acid residues at positions 31-35, a FR2 of a NBconstruct comprises the amino acids at positions 36-49, a CDR2 of a NBconstruct comprises the amino acid residues at positions 50-65, a FR3 ofa NB construct comprises the amino acid residues at positions 66-94, aCDR3 of a NB construct comprises the amino acid residues at positions95-102, and a FR4 of a NB construct comprises the amino acid residues atpositions 103-113. However, it is well known in the art for V_(H)domains and for V_(HH) domains that the total number of amino acidresidues in each of the CDR's may vary and therefore may not correspondto the total number of amino acid residues indicated by the Kabatnumbering. By way of example, for an inventive NB construct describedherein, the amino acid residues and their position in the NB constructfor their particular FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are asprovided in Table 4 herein. Other methods such as Chothia (Al-Lazikaniet al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)) may beused to define alternative versions of the particular CDRs, andcrystalographic determination of contact points and interaction sitesmay further inform one skilled in the art as to the extent of eachparticular CDR and/or FR region.

The Figures, Sequence Listing and the Experimental Part/Examples aregiven to further illustrate the invention and should not be interpretedor construed as limiting the scope of the invention and/or of theappended claims in any way, except if explicitly indicated otherwiseherein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the art referencescited herein.

In accordance with the terminology used in the art, the variable domainspresent in naturally occurring heavy chain antibodies will also bereferred to as “V_(HH) domains”, in order to distinguish them from theheavy chain variable domains that are present in conventional 4-chainantibodies (which will be referred to herein as “V_(H) domains”) andfrom the light chain variable domains that are present in conventional4-chain antibodies (which will be referred to herein as “V_(L)domains”). In general, V_(HH) domains have been “designed” by nature tofunctionally bind to an antigen without the presence of, and without anyinteraction with, a light chain variable domain.

As mentioned in the art references cited above, V_(HH) domains have anumber of unique structural characteristics and functional properties.These make isolated V_(HH) domains (as well as inventive compositionsbased thereon that share these structural characteristics and functionalproperties with the naturally occurring V_(HH) domains) and proteinscontaining the same highly advantageous for use as functionalantigen-binding domains or proteins. In particular, and without beinglimited thereto, V_(HH) domains and inventive compositions can functionas a single, relatively small, functional antigen-binding structuralunit, domain or protein. This distinguishes the V_(HH) domains from theV_(H) and V_(L) domains of conventional 4-chain antibodies, which bythemselves are generally not suited for practical application as singleantigen-binding proteins or domains, but need to be combined in someform or another to provide a functional antigen-binding unit (as in,e.g., conventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains, and ofinventive NB agents and compositions based thereon, as singleantigen-binding proteins or as antigen-binding domains (i.e., as part ofa larger protein or polypeptide) offers a number of significantadvantages over the use of conventional V_(H) and V_(L) domains, ScFv'sor conventional antibody fragments (such as Fab- or F(ab)₂-fragments).Advantages of a camelid V_(HH) variant of a NB construct are e.g., onlya single domain is required to bind an antigen with high affinity andwith high selectivity, so there is no need to manufacture or combinemultiple separate domains nor is there a need to assure that these twodomains are present in the right spacial conformation and configuration;V_(HH) domains can be expressed from a single transcript and require nopost-translational folding or modifications; V_(HH) domains can easilybe engineered into multivalent and multispecific formats (as furtherdiscussed herein); V_(HH) domains are highly soluble and do not have atendency to aggregate; V_(HH) domains are highly stable to heat, pH,proteases and other denaturing agents or conditions; V_(HH) domains areeasy and relatively cheap to prepare and to scale up for production;V_(HH) domains are relatively small (monomers are approximately 15 kDa,or 10 times smaller than a conventional IgG) compared to conventional4-chain antibodies and antigen-binding fragments thereof, and thereforeshow high(er) penetration into tissues (including but not limited tosolid tumors and other dense tissues) than such conventional 4-chainantibodies and antigen-binding fragments thereof; V_(HH) domains canshow so-called cavity-binding properties (inter alia due to theirextended CDR3 loop, compared to conventional V_(H) domains) and cantherefore also access targets and epitopes not accessible toconventional 4-chain antibodies and antigen-binding fragments thereof.

NB Constructs Against DR5

In a specific and preferred aspect, the invention provides NB agentsagainst DR5, and in particular NB agents against DR5 from a warm-bloodedanimal, and more in particular NB agents against DR5 from a mammal, andespecially NB agents against human DR5, such as provided in publicdatabases, e.g. in NCBI Protein database accession number BAA33723 (SEQID NO: 89); as well as proteins and/or polypeptides comprising at leastone such NB agent.

In particular, the invention provides NB agents against DR5, andproteins and/or polypeptides comprising the same, that have improvedtherapeutic and/or pharmacological properties and/or other advantageousproperties (such as, e.g., improved ease of preparation and/or reducedcosts of goods), compared to conventional antibodies against DR5, orfragments thereof, compared to constructs that could be based on suchconventional antibodies or antibody fragments (such as F_(ab)′fragments, F_((ab′)2) fragments, ScFv constructs, “diabodies” and othermultispecific constructs (see, e.g., the review by Holliger and Hudson,Nat Biotechnol. 2005 September; 23(9): 1126-36)), and also compared tothe so-called “dAb's” or similar (single) domain antibodies that may bederived from variable domains of conventional antibodies.

In one embodiment and as generally described herein for thetherapeutically useful amino acid sequences of the invention, the NBagents and variants thereof of the invention are in essentially isolatedform, or form part of a protein or polypeptide of the invention that maycomprise or essentially consist of one or more NB agents of theinvention. In one embodiment, the NB agent variant may further compriseone or more additional amino acid sequences. In one embodiment, the oneor more additional amino acid sequences are linked via one or moresuitable linkers. In one embodiment, and without limitation, the one ormore amino acid sequences of the invention may be used as a binding unitin such a protein or polypeptide, which may optionally contain one ormore further amino acid sequences that can serve as a binding unit(i.e., against one or more other targets than DR5), so as to provide amonovalent, multivalent or multispecific polypeptide of the invention,respectively. In one embodiment, such a protein or polypeptide maycomprise or essentially consist of one or more NB agents of theinvention and optionally one or more (other) polypeptide domains orepitope binding compositions (i.e., directed against other targets thanDR5), all optionally linked via one or more suitable linkers, so as toprovide a monovalent, multivalent or multispecific NB agent,respectively.

In one embodiment, the binding site for binding against DR5 is formed bythe CDR sequences. In one embodiment, an inventive composition may, inaddition to the at least one binding site for binding against DR5,contain one or more further binding sites for binding against otherantigens, proteins or targets. For methods and positions for introducingsuch second binding sites, reference is, e.g., made to Keck and Huston,Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130; WO06/07260 and the US provisional application by Ablynx N.V. entitled“Immunoglobulin domains with multiple binding sites” filed on Nov. 27,2006.

In one embodiment when the inventive amino acid sequences (or apolypeptide of the invention comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is directed against humanDR5. In one embodiment for veterinary purposes, the inventivecomposition is directed against DR5 from the species to be treated. Theinventive compositions herein may or may not be cross-reactive (i.e.,active against DR5 from two or more species of mammal, such as againsthuman DR5 and DR5 from at least one of the species of mammal mentionedherein). In one embodiment, a NB construct of the invention canspecifically bind to DR5 but does not bind to DR-4, TRAIL-R3 orTRAIL-R4.

In one embodiment, the compositions of the invention is generallydirected against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of DR5. In one embodiment,the inventive NB agents are directed against the extracellular domainportion of DR5.

In one specific embodiment, inventive compositions herein compete withthe natural ligand of DR5 in a competitive binding assay. In oneembodiment, an inventive NB agent competes with TRAIL for binding toDR5. In one embodiment, an inventive composition of the invention doesnot compete with TRAIL—for binding to DR5. In one embodiment, aninventive NB agent synergizes with TRAIL for binding to DR5.

In one non-limiting embodiment, the amino acid sequence and structure ofan inventive composition are comprised of four framework regions or“FR's” (or sometimes also referred to as “FW's”), which are referred toin the art and herein as “Framework region 1” or “FR1”; as “Frameworkregion 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Frameworkregion 4” or “FR4”, respectively; which framework regions areinterrupted by three complementary determining regions or “CDRs”, whichare referred to in the art as “Complementarity Determining Region 1” or“CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. In oneembodiment, the framework sequences and CDRs (and combinations thereof)are those present in the NB constructs of the invention as describedherein and especially in Table 4. Other suitable CDR sequences can beobtained by the methods described herein.

In one non-limiting embodiment, the CDR sequences of the invention aresuch that:

a) the NB constructs can bind to DR5 with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter(i.e., with an association constant (K_(A)) of 10⁵ to 10¹² liter/molesor more, and preferably 10⁷ to 10¹² liter/moles or more and morepreferably 10⁸ to 10¹² liter/moles); and/or such that

-   -   b) the NB constructs can bind to DR5 with a k_(on)-rate of        between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹; and/or        such that

c) the NB constructs can bind to DR5 with a k_(off) rate between 1 s-¹(t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible complexwith a t_(1/2) of multiple days), preferably between 10⁻² s-¹ and 10⁻⁶s-¹, more preferably between 10⁻³ s-¹ and 10⁻⁶ s-¹, such as between 10⁻⁴s-¹ and 10⁻⁶ s⁻¹.

In one embodiment, the CDR sequences herein are such that: a monovalentcomposition of the invention (or a polypeptide that contains only one NBconstruct of the invention) will bind to DR5 with an affinity less than100 nM, preferably less than 10 nM, more preferably less than 1 nM, suchas less than 500 pM.

The affinity of the NB agents of the invention against DR5 can bedetermined in a manner known per se, e.g., using the general techniquesfor measuring K_(D). K_(A), k_(off) or k_(on) mentioned herein, as wellas some of the specific assays described herein.

In one non-limiting aspect, the invention relates to a NB agent againstDR5, which consists of four framework regions (FR1 to FR4 respectively)and three complementarity determining regions (CDR1 to CDR3respectively). In one embodiment, the NB agent is a NB construct inwhich:

CDR1 is chosen from the group consisting of:

-   -   a) the amino acid sequences of SEQ ID NO's: 41 to 44;    -   b) amino acid sequences that have at least 90% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 41 to 44;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 41 to 44;

and/or

CDR2 is chosen from the group consisting of:

-   -   a) the amino acid sequences of SEQ ID NO's: 51 to 55;    -   b) amino acid sequences that have at least 90% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 51 to 55;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 51 to 55;

and/or

CDR3 is chosen from the group consisting of:

-   -   a) the amino acid sequences of SEQ ID NO's: 63 to 68;    -   b) amino acid sequences that have at least 90% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 63 to 68    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 63 to 68;        or any suitable fragment of such an amino acid sequence.

Of the NB agents of the invention, NB construct comprising one or moreof the CDR's explicitly listed above are particularly preferred; NBconstruct comprising two or more of the CDR's explicitly listed aboveare more particularly preferred; and NB construct comprising three ofthe CDR's explicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table 4 below, which lists the CDRsequences and framework sequences that are present in a number ofpreferred (but non-limiting) NB construct of the invention.

As will be clear to the skilled person, a combination of CDR1, CDR2 andCDR3 sequences that occur in the same clone (i.e., CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table 4, and especiallyas provided in SEQ ID NOS: 1-22, 26-40, 87-88, and 102-103) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table 4). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e.,CDR sequences and framework sequences that are mentioned on the sameline in Table 4, and especially as provided in context within SEQ IDNOS: 1-22, 26-40, 87-88, and 102-103)) will usually be preferred(although the invention in its broadest sense is not limited thereto,and also comprises other suitable combinations of the CDR sequences andframework sequences mentioned in Table 4, as well as combinations ofsuch CDR sequences and other suitable framework sequences.

In one embodiment, in the NB constructs that comprise the combinationsof CDR's mentioned in Table 4, each CDR can be replaced by a CDR chosenfrom the group consisting of amino acid sequences that have at least80%, preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with the mentioned CDR's.

Thus, in the NB constructs of the invention, at least one of the CDR1,CDR2 and CDR3 sequences present is suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable 4; or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% “sequenceidentity” with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table 4; and/or from the group consisting of theCDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1“amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table 4.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e., as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e.,as defined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e., as defined herein), respectively. In one embodiment, theCDR sequences are chosen such that the NB constructs of the inventionbind to DR5 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ valuethat is as provided herein.

In one embodiment of the NB constructs of the invention, at least theCDR3 sequence present is suitably chosen from the group consisting ofthe CDR3 sequences listed in Table 4 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table 4; and/or from the groupconsisting of the CDR3 sequences that have 3, or have 2 or have only 1amino acid difference(s) with at least one of the CDR3 sequences listedin Table 4.

In one embodiment of the NB constructs of the invention, at least two ofthe CDR1, CDR2 and CDR3 sequences present are suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table 4 or from the group consisting of CDR1, CDR2 and CDR3sequences, respectively, that have at least 80%, and/or at least 90%,and/or at least 95%, and/or at least 99% sequence identity with at leastone of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table4; and/or from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, that have 3, 2 or only 1 “amino aciddifference(s)” with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table 4.

In one embodiment of the NB constructs of the invention, at least theCDR3 sequence present is suitably chosen from the group consisting ofthe CDR3 sequences listed in Table 4 or from the group of CDR3 sequencesthat have at least 80%, and/or at least 90%, and/or at least 95%, and/orat least 99% sequence identity with at least one of the CDR3 sequenceslisted in Table 4, respectively; and at least one of the CDR1 and CDR2sequences present is suitably chosen from the group consisting of theCDR1 and CDR2 sequences, respectively, listed in Table 4 or from thegroup of CDR1 and CDR2 sequences, respectively, that have at least 80%,and/or at least 90%, and/or at least 95%, and/or at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table 4; and/or from the group consisting of the CDR1 and CDR2sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table 4.

In one embodiment of the NB constructs of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable 4.

In one embodiment, the combinations of CDR's listed in Table 4 (i.e.,those mentioned for the same construct in Table 4) are preferred. Thus,in one aspect, when a CDR in a NB construct of the invention is a CDRsequence mentioned in Table 4 or is suitably chosen from the group ofCDR sequences that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table 4; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table 4, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table 4 (i.e.,mentioned on the same line in Table 4) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in Table 4.

In one embodiment of a NB construct of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table 4.

According to one non-limiting aspect of a NB construct of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In one non-limiting aspect, the invention relates to a NB construct inwhich the CDR sequences have more than 80%, preferably more than 90%,more preferably more than 95%, such as at least 99% or more sequenceidentity with the CDR sequences of at least one of the amino acidsequences of SEQ ID NO's: 1-22, 26-40, 87-88, and 102-103.

Generally, NB agents with the above CDR sequences may be as furtherdescribed herein. In one embodiment the NB agents have frameworksequences that are also as further described herein. Thus, e.g., and asmentioned herein, such NB agents may be naturally occurring singledomain antibodies (from any suitable species), naturally occurringV_(HH) sequences (i.e., from a suitable species of Camelid) or syntheticor semi-synthetic amino acid sequences or NB agents, including but notlimited to partially humanized NB constructs or V_(HH) sequences, fullyhumanized NB constructs or V_(HH) sequences, camelized heavy chainvariable domain sequences, as well as NB agents that have been obtainedby the techniques mentioned herein.

Thus, in one non-limiting aspect, the invention relates to a humanizedNB agent, which consists of four framework regions (FR1 to FR4respectively) and three complementarity determining regions (CDR1 toCDR3 respectively), in which CDR1 to CDR3 are as defined herein and inwhich said humanized NB agent comprises at least one humanizingsubstitution, and in particular at least one humanizing substitution inat least one of its framework sequences.

In another preferred, but non-limiting aspect, the invention relates toa NB agent in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 1-22, 26-40,87-88, and 102-103. This degree of amino acid identity can, e.g., bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said NB agent and one or more of the sequencesof SEQ ID NO's: 1-22, 26-40, 87-88, and 102-103, in which the amino acidresidues that form the framework regions are disregarded. Such NB agentscan be as further described herein.

In one non-limiting aspect, the invention relates to an NB agent,including but not limited to a NB construct, with an amino acid sequencethat is chosen from the group consisting of SEQ ID NO's: 1-22, 26-40,87-88, and 102-103 or from the group consisting of from amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as at least 99% or more sequence identitywith at least one of the amino acid sequences of SEQ ID NO's: 1-22,26-40, 87-88, and 102-103.

Another non-limiting aspect of the invention relates to humanized orfurther humanized variants of the NB constructs of SEQ ID NO's: 1-22,26-40, 87-88, and 102-103 that comprise, compared to the correspondingnative sequence, at least one humanizing substitution, and in particularat least one humanizing substitution in at least one of its frameworksequences.

Monovalent and Multivalent Binding Polypeptides

The invention further relates to a compound that comprises oressentially consists of one or more polypeptides of the inventionagainst DR5, and more specifically against human DR5, and optionallycomprises one or more other groups, residues, moieties or binding units,wherein said compound is capable of enhancing apoptosis. In oneembodiment, some NB agents of the invention are proteins that compriseat least three, four, five or more monovalent binding polypeptidesagainst the DR5 receptor, such as NB agents of the invention directedagainst the DR5. As mentioned herein, in such multivalent polypeptidesof the invention, each monovalent binding polypeptide, such as a NBconstruct, may be directed against the same epitope on the DR5, oragainst different epitopes on the DR5. Some non-limiting examples ofsuch NB polypeptides of the invention are given in SEQ ID NO's: 1-22,26-40, 87-88, and 102-103.

Generally, proteins or polypeptides that comprise or essentially consistof a single binding polypeptide (such as a single DR5 binding subunit ofa NB agent of the invention) will be referred to herein as “monovalent”proteins or polypeptides or as “monovalent constructs”. Proteins andpolypeptides that comprise or essentially consist of two or more bindingpolypeptides (such as at least two joined NB agents of the invention orat least one NB agent of the invention and at least one other NB agentor polypeptide construct) will be referred to herein as “multivalent”proteins or polypeptides or as “multivalent constructs”, and these mayprovide certain advantages compared to the corresponding monovalentbinding polypeptides of the invention. Particular non-limiting examplesof such multivalent constructs are as provided herein.

Specific examples of multivalent NB agent include a dimer or divalentconstruct comprising two polypeptide subunits, a trimer or trivalent NBagent comprising three polypeptide subunits, a tetramer or tetravalentNB agent comprising four polypeptide subunits, a pentamer or pentavalentNB agent comprising five polypeptide subunits, a hexamer or hexavalentNB agent comprising six polypeptide subunits, or additional multimericvariants thereof.

In one non-limiting aspect, a trimeric NB agent of the inventioncomprises or essentially consists of three monovalent polypeptidesubunits of the invention. In one embodiment, the trimeric NB agentcomprises three identical DR5 binding monovalent polypeptide subunits ofthe invention, in which case the NB agent is a trimer that ismultivalent but monospecific. In one embodiment a trimeric NB agentcomprises or essentially consists of a first and/or second DR5 bindingsubunit and a second or third subunit, respectively, that is optionallya subunit that binds specifically to a different epitope (either on DR5or on another target), e.g., in which case the NB agent is a trimer thatis multivalent and multispecific.

In one non-limiting embodiment, a NB agent comprises or essentiallyconsists of at least four monovalent DR5 binding polypeptide subunits ofthe invention. Such a tetrameric NB agent of the invention may bemonospecific or may be converted to a multispecific construct byoptionally joining additional subunits that bind specifically to thesame or a different DR5 epitope from the first and/or to a target otherthan DR5.

In one non-limiting embodiment, a NB agent comprises or essentiallyconsists of at least five monovalent DR5 binding polypeptide subunits ofthe invention. Such a pentameric NB agent of the invention may bemonospecific or may be converted to a multispecific construct byoptionally joining additional subunits that bind specifically to adifferent DR5 epitope from the first and/or to a target other than DR5.

In one non-limiting embodiment, a NB agent comprises or essentiallyconsists of six, eight or ten monovalent DR5 binding polypeptidesubunits of the invention. Such a multimeric NB agent of the inventionmay be monospecific or may be converted to a multispecific construct byoptionally joining additional subunits that bind specifically to adifferent DR5 epitope from the first and/or to a target other than DR5.

In one embodiment, such multimeric NB agents, whether monospecific ormultispecific, provide certain advantages compared to a protein orpolypeptide comprising or essentially consisting of a monomeric NB agentof the invention, In one embodiment, advantages include, but are notlimited to, a much improved avidity for DR5, e.g., for human DR5. Somespecific, but non-limiting examples of multimeric NB agents are the NBconstructs of SEQ ID NO's: 6 to 22, 27-29, 31-33 and 88.

In one non-limiting aspect, a polypeptide of the invention comprises oressentially consists of at least one NB agent of the invention,optionally one or more further NB agents, and at least one other aminoacid sequence (such as a protein or polypeptide) that confers at leastone desired property to the NB agent of the invention and/or to theresulting fusion protein. Again, such fusion proteins may providecertain advantages compared to the corresponding monovalent NB agent ofthe invention. Some non-limiting examples of such amino acid sequencesand of such fusion constructs will become clear from the furtherdescription herein.

In one embodiment, it is possible to combine two or more of the aboveaspects, e.g., to provide a trivalent bispecific construct comprisingtwo NB agents of the invention and one other NB agent, and optionallyone or more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more binding polypeptides againstDR5 and/or other amino acid sequences may be operably linked to eachother and/or suitably linked to each other via one or more linkersequences. Some suitable but non-limiting examples of such linkers willbecome clear from the further description herein.

In one specific aspect, the multivalent compound of the inventioncomprises at least three, four, five or more monovalent bindingpolypeptides (i.e., subunits) and has an IC₅₀ less than 100 nM,preferably less than 10 nM, more preferably less than 1 nM, even morepreferably less than 100 pM, e.g., below 10 pM as measured, e.g., inColo205 or Jurkat cell survival assay. Other cancer cell lines can beused for determining IC₅₀, e.g., such as mentioned in the Examples.Furthermore, possible cancer cell lines that may be used in cellsurvival assays include, but are not limited to, e.g., Jurkat, Molt4,Colo205, BxPC3, T24, Panc-1, M30, H226, H2122, H2052, and MiaPaCa-2.

In one embodiment, the multivalent compound of the invention are atleast 10 fold, preferably at least 100 fold more potent in a tumor cellline than in a non-tumor cell line and measured in a cell survival assaysuch as Colo205 or Jurkat cell survival assay.

In one specific aspect of the invention, a NB agent of the invention ora compound, construct or polypeptide of the invention comprising atleast one NB agent of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somenon-limiting examples of such NB agents, compounds and polypeptides willbecome clear to the skilled person based on the further disclosureherein, and, e.g., comprise NB agent sequences or polypeptides of theinvention that have been chemically modified to increase the half-lifethereof (for example, by means of pegylation); amino acid sequences ofthe invention that comprise at least one additional binding site forbinding to a serum protein (such as serum albumin. or polypeptides ofthe invention that comprise at least one NB agent of the invention thatis linked to at least one moiety (and in particular at least one aminoacid sequence) that increases the half-life of the NB agent of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or amino acid sequences will become clearto the skilled person based on the further disclosure herein; and, e.g.,include, without limitation, polypeptides in which the one or more NBagents of the invention are suitable linked to one or more serumproteins or fragments thereof (such as serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, e.g., NB agents, camelid V_(HH) constructs or(single) domain antibodies that can bind to serum proteins such as serumalbumin, serum immunoglobulins such as IgG, or transferrine);polypeptides in which a NB agent of the invention is linked to an Fcportion (such as a human Fc) or a suitable part or fragment thereof; orpolypeptides in which the one or more NB agents of the invention aresuitable linked to one or more small proteins or peptides that can bindto serum proteins (such as, without limitation, the proteins andpeptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to WO2006/122787, WO 2008/028977, WO 2008/043821, WO 2008/068280 and WO2009/127691 filed by Ablynx N.V.

Again, as will be clear to the skilled person, such NB agents maycontain one or more additional groups, residues, moieties or bindingunits, such as one or more further amino acid sequences and inparticular one or more additional NB agents (i.e., not directed againstDR5), so as to provide a di-, tri- or higher multispecific NB agentconstruct.

Generally, the NB agents of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, e.g., at least 10 times or more than 20times, greater than the half-life of the corresponding amino acidsequence of the invention per se. For example, the NB agents, compounds,constructs or polypeptides of the invention with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such NBagents, compound, constructs or polypeptides of the invention exhibit aserum half-life in human of at least about 12 hours, preferably at least24 hours, more preferably at least 48 hours, even more preferably atleast 72 hours or more. For example, compounds or polypeptides of theinvention may have a half-life of at least 5 days (such as about 5 to 10days), preferably at least 9 days (such as about 9 to 14 days), morepreferably at least about 10 days (such as about 10 to 15 days), or atleast about 11 days (such as about 11 to 16 days), more preferably atleast about 12 days (such as about 12 to 18 days or more), or more than14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the inventioncomprises one or more (such as two or preferably one) NB agents of theinvention linked (optionally via one or more suitable linker sequences)to one or more (such as two and preferably one) amino acid sequencesthat allow the resulting polypeptide of the invention to cross the bloodbrain barrier. In particular, said one or more amino acid sequences thatallow the resulting polypeptides of the invention to cross the bloodbrain barrier may be one or more (such as two and preferably one)NANOBODIES™, such as the NANOBODIES™ described in WO 02/057445, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are particular examples.

In one embodiment, polypeptides comprising one or more NB agents of theinvention are such that they:

-   -   a) bind to DR5 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e., with an association constant (K_(A)) of 10⁵        to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   b) bind to DR5 with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between        10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   c) bind to DR5 with a k_(off) rate between 1 s⁻¹        (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

In one embodiment, a polypeptide that contains only one amino acidsequence of the invention is such that it will bind to DR5 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 500 pM. In this respect, it will beclear to the skilled person that a polypeptide that contains two or moreNB agents of the invention may bind to DR5 with an increased avidity,compared to a polypeptide that contains only one amino acid sequence ofthe invention.

Other polypeptides according to this preferred aspect of the inventionmay, e.g., be chosen from the group consisting of amino acid sequencesthat have more than 80%, preferably more than 90%, more preferably morethan 95%, such as at least 99% or more “sequence identity” with one ormore of the amino acid sequences of SEQ ID NO's: 1-22, 26-40 and 87-88,in which the NB agents comprised within said amino acid sequences arepreferably as further defined herein.

Nucleic Acids, Host Cells and Method for Generating NB Constructs of theInvention

Another aspect of this invention relates to a nucleic acid that encodesa NB agent of the invention or a polypeptide of the invention comprisingthe same. Again, as generally described herein for the nucleic acids ofthe invention, such a nucleic acid may be in the form of a geneticconstruct, as provided herein.

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing a NB agent of the inventionand/or a polypeptide of the invention comprising the same; and/or thatcontains a nucleic acid of the invention. Some preferred butnon-limiting examples of such hosts or host cells will become clear fromthe further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one NB agent of the invention, atleast one polypeptide of the invention and/or at least one nucleic acidof the invention, and optionally one or more further components of suchcompositions known per se, i.e., depending on the intended use of thecomposition. Such a product or composition may, e.g., be apharmaceutical composition (as described herein), a veterinarycomposition or a product or composition for diagnostic use (as alsodescribed herein). Some preferred but non-limiting examples of suchproducts or compositions will become clear from the further descriptionherein.

The invention further relates to methods for preparing or generating theNB agents, polypeptides, nucleic acids, host cells, products andcompositions described herein. Some preferred but non-limiting examplesof such methods will become clear from the further description herein.

The invention further relates to applications and uses of the NB agents,polypeptides, nucleic acids, host cells, products and compositionsdescribed herein, as well as to methods for the prevention and/ortreatment for diseases and disorders associated with DR5. Particularnon-limiting applications and uses will become clear from the furtherdescription herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description hereinbelow.

Generally, it should be noted that the term “NB agent” as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the NB agents of the invention can generally beobtained by any of the techniques (1) to (8) mentioned on pages 61 and62 of WO 08/020079, or any other suitable technique known per se. Onepreferred class of NB agents corresponds to the V_(HH) or V_(H) domainsof naturally occurring heavy chain antibodies directed against DR5. Asfurther described herein, V_(HH) sequences can generally be generated orobtained by suitably immunizing a species of Camelid with DR5 (i.e., soas to raise an immune response and/or heavy chain antibodies directedagainst DR5), by obtaining a suitable biological sample from saidCamelid (such as a blood sample, serum sample or sample of B-cells), andby generating V_(HH) sequences directed against DR5, starting from saidsample, using any suitable technique known per se. Such techniques willbe clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against DR5, canbe obtained from naïve libraries of Camelid V_(HH) sequences, e.g., byscreening such a library using DR5, or at least one part, fragment,antigenic determinant or epitope thereof using one or more screeningtechniques known per se. Such libraries and techniques are, e.g.,described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694.Alternatively, improved synthetic or semi-synthetic libraries derivedfrom naïve V_(HH) libraries may be used, such as V_(HH) librariesobtained from naïve V_(HH) libraries by techniques such as randommutagenesis and/or CDR shuffling, as, e.g., described in WO 00/43507.

Thus, in another aspect, the invention relates to a method forgenerating NB agents that are directed against DR5. In one aspect, saidmethod at least comprises the steps of:

-   -   a) providing a set, collection or library of NB agent sequences;    -   b) screening said set, collection or library of NB agent        sequences for NB agent sequences that can bind to and/or have        affinity for DR5;

and

-   -   c) isolating the amino acid sequence(s) that can bind to and/or        have affinity for DR5.

In such a method, the set, collection or library of NB agent sequencesmay be a naïve set, collection or library of NB agent sequences; asynthetic or semi-synthetic set, collection or library of NB agentsequences; and/or a set, collection or library of NB agent sequencesthat have been subjected to affinity maturation.

In a particular aspect of this method, the set, collection or library ofNB agent sequences may be an immune set, collection or library of NBagent sequences, and in particular an immune set, collection or libraryof V_(HH) sequences, that have been derived from a species of Camelidthat has been suitably immunized with DR5 or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of V_(HH) sequencesmay be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) sequences will be clear tothe person skilled in the art, e.g., on the basis of the furtherdisclosure herein. Reference is also made to WO 03/054016 and to thereview by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In one embodiment, the method for generating V_(HH) sequences comprisesat least the steps of:

-   -   a) providing a collection or sample of cells derived from a        species of Camelid that express immunoglobulin sequences;    -   b) screening said collection or sample of cells for (1) cells        that express an immunoglobulin sequence that can bind to and/or        have affinity for DR5; and (2) cells that express heavy chain        antibodies, in which substeps (1) and (2) can be performed        essentially as a single screening step or in any suitable order        as two separate screening steps, so as to provide at least one        cell that expresses a heavy chain antibody that can bind to        and/or has affinity for DR5;        and    -   c) either (1) isolating from said cell the V_(HH) sequence        present in said heavy chain antibody; or (2) isolating from said        cell a nucleic acid sequence that encodes the V_(HH) sequence        present in said heavy chain antibody, followed by expressing        said V_(HH) domain.

In the method according to this aspect, the collection or sample ofcells may, e.g., be a collection or sample of B-cells. Also, in thismethod, the sample of cells may be derived from a Camelid that has beensuitably immunized with DR5 or a suitable antigenic determinant basedthereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is, e.g., made to EP 0 542 810,WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) ispreferably performed using a flow cytometry technique such as FACS. Forthis, reference is, e.g., made to Lieby et al., Blood, Vol. 97, No. 12,3820. See, e.g., the so-called “Nanoclone™” technique described inInternational application WO 06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against DR5 may comprise at least the steps of:

-   -   a) providing a set, collection or library of nucleic acid        sequences encoding heavy chain antibodies or V_(HH) sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode a heavy chain        antibody or a V_(HH) sequence that can bind to and/or has        affinity for DR5; and    -   c) isolating said nucleic acid sequence, followed by expressing        the V_(HH) sequence present in said heavy chain antibody or by        expressing said NB construct sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or V_(HH) sequences may, e.g.,be a set, collection or library of nucleic acid sequences encoding anaïve set, collection or library of heavy chain antibodies or V_(HH)sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofV_(HH) sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of V_(HH) sequences thathave been subjected to affinity maturation.

In one embodiment of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withDR5 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one aspect, said antigenic determinant may bean extracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, e.g., on the basis of the further disclosure herein. Reference isalso made to WO 03/054016 and to Hoogenboom in Nature Biotechnology, 23,9, 1105-1116 (2005).

In one embodiment, the screening step of the methods described hereincan also be performed as a selection step. Accordingly the term“screening” as used in the present description can comprise selection,screening or any suitable combination of selection and/or screeningtechniques. Also, when a set, collection or library of sequences isused, it may contain any suitable number of sequences, such as 1, 2, 3or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ ormore sequences.

In one embodiment, one or more or all of the sequences in the above set,collection or library of amino acid sequences may be obtained or definedby rational, or semi-empirical approaches such as computer modelingtechniques or biostatics or data mining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library)), or any other source of diverse sequences (asdescribed, e.g., in Hoogenboom et al, Nat Biotechnol 23: 1105, 2005 andBinz et al, Nat Biotechnol 2005, 23: 1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences, V_(H) sequences,or some other variant of NB agent sequences directed against DR5involves suitably immunizing a transgenic mammal that is capable ofexpressing heavy chain antibodies (i.e., so as to raise an immuneresponse and/or heavy chain antibodies directed against DR5), obtaininga suitable biological sample from said transgenic mammal that contains(nucleic acid sequences encoding) said sequences (such as a bloodsample, serum sample or sample of B-cells), and then generating NB agentsequences directed against DR5, starting from said sample, using anysuitable technique known per se (such as any of the methods describedherein or a hybridoma technique). For example, for this purpose, theheavy chain antibody-expressing mice and the further methods andtechniques described in WO 02/085945, WO 04/049794 and WO 06/008548 andJanssen's et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can be used. For example, such heavy chain antibody expressingmice can express heavy chain antibodies with any suitable (single)variable domain, such as (single) variable domains from natural sources(e.g. human (single) variable domains, Camelid (single) variable domainsor shark (single) variable domains), as well as, e.g., synthetic orsemi-synthetic (single) variable domains.

In one embodiment the invention relates to the NB agent sequences thatare obtained by the above methods, or alternatively by a method thatcomprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid NB agent sequence; and of expressing or synthesizing said NB agentsequence in a manner known per se, such as by expression in a suitablehost cell or host organism or by chemical synthesis.

In one embodiment the invention provides a class of NB agents thatcomprises camelid V_(HH) constructs with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring llamaV_(HH) domain, but that has been “humanized”, i.e., by replacing one ormore amino acid residues in the amino acid sequence of said naturallyoccurring V_(HH) sequence (and in particular in the framework sequences,but also optionally in one or more CDR) by one or more of the amino acidresidues that occur at the corresponding position(s) in a V_(H) domainfrom a conventional four-chain antibody from a human being, e.g. asindicated above, and e.g., as further described on, and using thetechniques mentioned on, page 63 of WO 08/020079. In one embodiment theinvention provides a class of NB agents that comprises an amino acidsequence that corresponds to the amino acid sequence of a naturallyoccurring V_(H) domain, but that has been “camelized”, i.e., byreplacing one or more amino acid residues in the amino acid sequence ofa naturally occurring V_(H) domain from a conventional four-chainantibody by one or more of the amino acid residues that occur at thecorresponding position(s) in V_(HH) domain of a heavy chain antibody,e.g., as further described on, and using the techniques mentioned on,page 63 of WO 08/020079.

Other suitable methods and techniques for obtaining the NB agents of theinvention (including polypeptides and/or nucleic acids encoding thesame), starting from naturally occurring V_(H) sequences or V_(HH)sequences, will be clear to the skilled person, and may, e.g., includethe techniques that are mentioned on page 64 of WO 08/020079.

As mentioned herein, NB agents may in particular be characterized by thepresence of one or more “Hallmark residues” (as described in WO2008/020079, pages 65-98) in one or more of the framework sequences.

In one embodiment NB agents may, e.g., be V_(HH) sequences or may behumanized or further humanized NB constructs. When the NB agentsequences are V_(HH) sequences, they may be suitably humanized. When theNB agents are partially humanized NB constructs, they may optionally befurther suitably humanized. In the above NB agents, one or more of thefurther Hallmark residues are preferably as described (for example, whenthey are V_(HH) sequences or partially or fully humanized NBconstructs).

The NB agents herein may, e.g., be V_(HH) sequences or may be humanizedor humaneered. When the above NB agents sequences are V_(HH) sequences,they may be suitably humanized, as provided herein. When the NB agentsare partially humanized, they may optionally be further suitablyhumanized, as described herein. One nonlimiting and exemplary method ofhumanization is provided in the examples, but other methods known in theart may be contemplated.

In one embodiment of the above NB agents, one or more of the (further)Hallmark residues are as described herein (for example, when they arecamelid V_(HH) sequences or partially or fully humanized V_(HH)constructs).

In one non-limiting aspect, the invention relates to a NB agents asdescribed above, in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as at least 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 1-22, 26-40, 87-88, and 102-103. This degree of amino acididentity can, e.g., be determined by determining the degree of aminoacid identity (in a manner described herein) between said NB agents andone or more of the sequences of SEQ ID NO's: 1-22, 26-40, 87-88, and102-103, in which the amino acid residues that form the frameworkregions are disregarded. Such NB agents can be modified as furtherdescribed herein.

As already mentioned herein, another preferred but non-limiting aspectof the invention relates to a NB agent with an amino acid sequence thatis chosen from the group consisting of SEQ ID NO's: 1-22, 26-40, 87-88,and 102-103 or from the group consisting of from amino acid sequencesthat have more than 80%, preferably more than 90%, more preferably morethan 95%, such as at least 99% or more sequence identity with at leastone of the amino acid sequences of SEQ ID NO's: 1-22, 26-40, 87-88, and102-103.

In particular embodiments of the above NB agents:

-   -   i) any amino acid substitution (when it is not a humanizing        substitution as defined herein) is preferably, and compared to        the corresponding amino acid sequence of SEQ ID NO's: 1-22,        26-40 and 87-88, a conservative amino acid substitution;        and/or:    -   ii) its amino acid sequence preferably contains either only        amino acid substitutions, or otherwise preferably no more than        5, preferably no more than 3, and more preferably only 1 or 2        amino acid deletions or insertions, compared to the        corresponding amino acid sequence of SEQ ID NO's: 1-22, 26-40        and 87-88;        and/or    -   iii) the CDR's may be CDR's that are derived by means of        affinity maturation, e.g., starting from the CDR's of to the        corresponding amino acid sequence of SEQ ID NO's: 1-22, 26-40        and 87-88.

Preferably, the CDR sequences and FR sequences in the NB agents of theinvention are such that the NB agents of the invention (and polypeptidescomprising the same):

-   -   a) bind to a DR5, e.g., human DR5, with a dissociation constant        (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably        10⁻⁷ to 10⁻¹² moles/liter or less and more preferably 10⁻⁸ to        10⁻¹² moles/liter (i.e., with an association constant (K_(A)) of        10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to 10¹²        liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that they:    -   b) bind to a DR5, e.g., human DR5, with a k_(on)-rate of between        10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹ preferably between 10³ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹,        such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   c) bind to a DR5, e.g., human DR5, with a k_(off) rate between 1        s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near irreversible        complex with a t_(1/2) of multiple days), preferably between        10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶        s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

In one embodiment, CDR sequences and FR sequences present in the NBagents of the invention are such that the NB agents of the inventionwill bind to a DR5, e.g., human DR5, with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

In one non-limiting aspect of the invention, a NB agent may be asprovided herein, but with the proviso that it has at least “one aminoacid difference” in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring human V_(H)domain, and in particular compared to the corresponding framework regionof DP-47. More specifically, according to one non-limiting aspect of theinvention, a NB agent may be as provided herein, but with the provisothat it has at least “one amino acid difference” at at least one of theHallmark residues (including those at positions 108, 103 and/or 45)compared to the corresponding framework region of a naturally occurringhuman V_(H) domain, and in particular compared to the correspondingframework region of DP-47. Usually, a NB agent will have at least onesuch amino acid difference with a naturally occurring V_(H) domain in atleast one of FR2 and/or FR4, and in particular at at least one of theHallmark residues in FR2 and/or FR4 (again, including those at positions108, 103 and/or 45).

In one embodiment, a humanized NB agent of the invention may be asprovided herein, but with the proviso that it has at least “one aminoacid difference” in at least one of the framework regions compared tothe corresponding framework region of a naturally occurring V_(HH)domain. More specifically, according to one non-limiting aspect of theinvention, a humanized NB agent may be as provided herein, but with theproviso that it has at least “one amino acid difference” at at least oneof the Hallmark residues (including those at positions 108, 103 and/or45) compared to the corresponding framework region of a naturallyoccurring V_(HH) domain. Usually, a humanized NB agent will have atleast one such amino acid difference with a naturally occurring V_(HH)domain in at least one of FR2 and/or FR4, and in particular at at leastone of the Hallmark residues in FR2 and/or FR4 (again, including thoseat positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is within the scope ofthe invention to use natural or synthetic analogs, mutants, variants,alleles, homologs and orthologs (herein collectively referred to as“analogs”) of the NB agents of the invention as provided herein, and inparticular analogs of the NB constructs of SEQ ID NO's 1-22, 26-40,87-88, and 102-103 and of SEQ ID NOS: 96-99. Thus, according to oneaspect of the invention, the term “NB agents of the invention” and theterm “NB constructs of the invention” in its broadest sense also coverssuch analogs.

Generally, in such analogs, one or more amino acid residues may havebeen replaced, deleted and/or added, compared to the NB agents of theinvention as provided herein. Such substitutions, insertions ordeletions may be made in one or more of the framework regions and/or inone or more of the CDR's. When such substitutions, insertions ordeletions are made in one or more of the framework regions, they may bemade at one or more of the Hallmark residues and/or at one or more ofthe other positions in the framework residues, although substitutions,insertions or deletions at the Hallmark residues are generally lesspreferred (unless these are suitable humanizing substitutions asdescribed herein).

By means of non-limiting examples, a substitution may, e.g., be aconservative substitution (as described herein) and/or an amino acidresidue may be replaced by another amino acid residue that naturallyoccurs at the same position in another V_(HH) domain (see e.g., WO2008/020079 for non-limiting examples of such substitutions), althoughthe invention is generally not limited thereto. Thus, any one or moresubstitutions, deletions or insertions, or any combination thereof, thateither improve the properties of the NB agents of the invention or thatat least do not detract too much from the desired properties or from thebalance or combination of desired properties of the NB agents of theinvention (i.e., to the extent that the NB agent is no longer suited forits intended use) are included within the scope of the invention. Askilled person will generally be able to determine and select suitablesubstitutions, deletions or insertions, or suitable combinations ofthereof, based on the disclosure herein and optionally after a limiteddegree of routine experimentation, which may, e.g., involve introducinga limited number of possible substitutions and determining theirinfluence on the properties of the NB agents thus obtained.

In one aspect, depending on the host organism used to express the NBagent (i.e., a polynucleotide or polypeptide of the invention), suchdeletions and/or substitutions may be designed in such a way that one ormore sites for post-translational modification (such as one or moreglycosylation sites) are removed, as will be within the ability of theperson skilled in the art. Alternatively, substitutions or insertionsmay be designed so as to introduce one or more sites for attachment offunctional groups (as described herein), e.g., to allow site-specificpegylation (again as described herein).

As provided in WO 2008/020079 regarding possible amino acidsubstitutions and as presented above, some amino acid residues in theframework regions are more conserved than others. In one aspect,although the invention in its broadest sense is not limited thereto, anysubstitutions, deletions or insertions are preferably made at positionsthat are less conserved. In one nonlimiting aspect, amino acidsubstitutions are preferred over amino acid deletions or insertions.

In one nonlimiting aspect, the analogs are such that they can bind toDR5 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as provided herein for the NB agentsof the invention.

In one nonlimiting aspect, the analogs are such that they retain thefavorable properties of the NB agents, as described herein.

In one embodiment, the analogs have a degree of sequence identity of atleast 70%, preferably at least 80%, more preferably at least 90%, suchas at least 95% or 99% or more; and/or preferably have at most 20,preferably at most 10, even more preferably at most 5, such as 4, 3, 2or only 1 amino acid difference, with one of the NB constructs of SEQ IDNOs: 1-22, 26-40, 87-88, and 102-103.

In a nonlimiting aspect, the framework sequences and CDR's of theanalogs will have (a) a Q at position 108; and/or (b) a charged aminoacid or a cysteine residue at position 45 and preferably an E atposition 44, and more preferably E at position 44 and R at position 45;and/or (c) P, R or S at position 103.

In one nonlimiting aspect, one class of analogs of the NB agents of theinvention comprise NB agents that have been humanized (i.e., compared tothe sequence of a naturally occurring NB agents of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, e.g., from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a NB agent and the sequence of anaturally occurring human V_(H) domain.

In one nonlimiting aspect, the humanizing substitutions are chosen suchthat the resulting humanized NB agents still retain the favorableproperties of NB agents as provided herein, and further such that theyare as described for analogs in the preceding paragraphs. A skilledperson will generally be able to determine and select suitablehumanizing substitutions or suitable combinations of humanizingsubstitutions, based on the disclosure herein and optionally after alimited degree of routine experimentation, which may, e.g., involveintroducing a limited number of possible humanizing substitutions anddetermining their influence on the properties of the NB agents thusobtained.

In one nonlimiting aspect, as a result of humanization, the NB agents ofthe invention may become more “human-like”, while still retaining thefavorable properties of the NB agents of the invention as describedherein. As a result, such humanized NB agents may have severaladvantages, such as a reduced immunogenicity, compared to thecorresponding naturally occurring V_(HH) domains. Again, based on thedisclosure herein and optionally after a limited degree of routineexperimentation, the skilled person will be able to select humanizingsubstitutions or suitable combinations of humanizing substitutions whichoptimize or achieve a desired or suitable balance between the favorableproperties provided by the humanizing substitutions on the one hand andthe favorable properties of naturally occurring V_(HH) domains on theother hand.

The NB agents of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues or at oneor more other framework residues (i.e., non-Hallmark residues) or anysuitable combination thereof. One preferred humanizing substitution forNB agents of the “P,R,S-103 group” or the “KERE group” is Q108 intoL108. NB agents of the “GLEW class” may also be humanized by a Q108 intoL108 substitution, provided at least one of the other Hallmark residuescontains a camelid (camelizing) substitution. For example, as mentionedherein, one particularly preferred class of humanized NB agents has GLEWor a GLEW-like sequence at positions 44-47; P, R or S (and in particularR) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se, e.g., using one ormore of the techniques mentioned on pages 103 and 104 of WO 08/020079.

In one nonlimiting aspect, one class of analogs of the NB agents of theinvention comprise NB agents that have been humanized (i.e., compared tothe sequence of a naturally occurring NB agent of the invention). Asmentioned in the background art cited herein, such humanizationgenerally involves replacing one or more amino acid residues in thesequence of a naturally occurring V_(HH) with the amino acid residuesthat occur at the same position in a human V_(H) domain, such as a humanV_(H)3 domain. Examples of possible humanizing substitutions orcombinations of humanizing substitutions will be clear to the skilledperson, e.g., from the Tables herein, from the possible humanizingsubstitutions mentioned in the background art cited herein, and/or froma comparison between the sequence of a NB agent and the sequence of anaturally occurring human V_(H) domain.

In one nonlimiting aspect, the humanizing substitutions are chosen suchthat the resulting humanized NB agents still retain the favorableproperties of NB agents as provided herein, and more preferably suchthat they are as described for analogs in the preceding paragraphs. Askilled person will generally be able to determine and select suitablehumanizing substitutions or suitable combinations of humanizingsubstitutions, based on the disclosure herein and optionally after alimited degree of routine experimentation, which may, e.g., involveintroducing a limited number of possible humanizing substitutions anddetermining their influence on the properties of the NB agents thusobtained.

Generally, as a result of humanization, the NB agents of the inventionmay become more “human-like”, while still retaining the favorableproperties of the NB agents of the invention as described herein. As aresult, such humanized NB agents may have several advantages, such as areduced immunogenicity, compared to the corresponding naturallyoccurring V_(HH) domains. Again, based on the disclosure herein andoptionally after a limited degree of routine experimentation, theskilled person will be able to select humanizing substitutions orsuitable combinations of humanizing substitutions which optimize orachieve a desired or suitable balance between the favorable propertiesprovided by the humanizing substitutions on the one hand and thefavorable properties of naturally occurring V_(HH) domains on the otherhand.

The NB agents of the invention may be suitably humanized at anyframework residue(s), such as at one or more Hallmark residues (asdefined in WO 2008/020079) or at one or more other framework residues(i.e., non-Hallmark residues) or any suitable combination thereof.

The humanized and other analogs, and nucleic acid sequences encoding thesame, can be provided in any manner known per se. For example, theanalogs can be obtained by providing a nucleic acid that encodes anaturally occurring V_(HH) domain, changing the codons for the one ormore amino acid residues that are to be substituted into the codons forthe corresponding desired amino acid residues (e.g. by site-directedmutagenesis or by PCR using suitable mismatch primers), expressing thenucleic acid/nucleotide sequence thus obtained in a suitable host orexpression system; and optionally isolating and/or purifying the analogthus obtained to provide said analog in essentially isolated form (e.g.as further described herein).

This can generally be performed using methods and techniques known perse, which will be clear to the skilled person, e.g., from the handbooksand references cited herein, the background art cited herein and/or fromthe further description herein. Alternatively, a nucleic acid encodingthe desired analog can be synthesized in a manner known per se (forexample using an automated apparatus for synthesizing nucleic acidsequences with a predefined amino acid sequence) and can then beexpressed as described herein. Yet another technique may involvecombining one or more naturally occurring and/or synthetic nucleic acidsequences each encoding a part of the desired analog, and thenexpressing the combined nucleic acid sequence as described herein. Also,the analogs can be provided using chemical synthesis of the pertinentamino acid sequence using techniques for peptide synthesis known per se,such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that theNB agents of the invention (including their analogs) can be designedand/or prepared starting from human V_(H) sequences (i.e., amino acidsequences or the corresponding nucleotide sequences), such as, e.g.,from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e., byintroducing one or more camelizing substitutions (i.e., changing one ormore amino acid residues in the amino acid sequence of said human V_(H)domain into the amino acid residues that occur at the correspondingposition in a V_(HH) domain), so as to provide the sequence of a NBagent of the invention and/or so as to confer the favorable propertiesof a NB agent to the sequence thus obtained. Again, this can generallybe performed using the various methods and techniques referred to in theprevious paragraph, using an amino acid sequence and/or nucleotidesequence for a human V_(H) domain as a starting point.

As mentioned there, it will be clear to the skilled person that the NBagents of the invention (including their analogs) can be designed and/orprepared starting from human V_(H) sequences (i.e., amino acid sequencesor the corresponding nucleotide sequences), such as, e.g., from humanV_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e., by introducing oneor more camelizing substitutions (i.e., changing one or more amino acidresidues in the amino acid sequence of said human V_(H) domain into theamino acid residues that occur at the corresponding position in a V_(HH)domain), so as to provide the sequence of a NB agent of the inventionand/or so as to confer the favorable properties of a NB agents to thesequence thus obtained. Again, this can generally be performed using thevarious methods and techniques referred to in the previous paragraph,using an amino acid sequence and/or nucleotide sequence for a humanV_(H) domain as a starting point.

Some non-limiting camelizing substitutions can be derived from WO2008/020079. It will be clear that camelizing substitutions at one ormore of the Hallmark residues will generally have a greater influence onthe desired properties than substitutions at one or more of the otheramino acid positions, although both and any suitable combination thereofare included within the scope of the invention. For example, it ispossible to introduce one or more camelizing substitutions that alreadyconfer at least some the desired properties, and then to introducefurther camelizing substitutions that either further improve saidproperties and/or confer additional favorable properties. Again, theskilled person will generally be able to determine and select suitablecamelizing substitutions or suitable combinations of camelizingsubstitutions, based on the disclosure herein and optionally after alimited degree of routine experimentation, which may, e.g., involveintroducing a limited number of possible camelizing substitutions anddetermining whether the favorable properties of NB agents are obtainedor improved (i.e., compared to the original V_(H) domain).

In one nonlimiting aspect, such camelizing substitutions are such thatthe resulting an amino acid sequence at least contains (a) a Q atposition 108; and/or (b) a charged amino acid or a cysteine residue atposition 45 and preferably also an E at position 44, and more preferablyE at position 44 and R at position 45; and/or (c) P, R or S at position103; and optionally one or more further camelizing substitutions. In onenonlimiting aspect, the camelizing substitutions are such that theyresult in a NB agent of the invention and/or in an analog thereof, suchas in a humanized analog and/or preferably in an analog that is asprovided in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within thescope of the invention to use parts or fragments, or combinations of twoor more parts or fragments, of the NB agents of the invention asprovided herein, and in particular parts or fragments of the NB agentsof SEQ ID NO's: 1-22, 26-40, 87-88, and 102-103 and of SEQ ID NOS:

96-99. Thus, according to one aspect of the invention, the term “NBagents of the invention” in its broadest sense also covers such parts orfragments.

Generally, such parts or fragments of the NB agents of the invention(including analogs thereof) have amino acid sequences in which, comparedto the amino acid sequence of the corresponding full length NB agent ofthe invention (or analog thereof), one or more of the amino acidresidues at the N-terminal end, one or more amino acid residues at theC-terminal end, one or more contiguous internal amino acid residues, orany combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to a DR5,e.g., human DR5 with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein) that is as provided herein for the NBagents of the invention.

Any part or fragment is preferably such that it comprises at least 10contiguous amino acid residues, preferably at least 20 contiguous aminoacid residues, more preferably at least 30 contiguous amino acidresidues, such as at least 40 contiguous amino acid residues, of theamino acid sequence of the corresponding full length NB agent of theinvention.

Also, any part or fragment is such preferably that it comprises at leastone of CDR1, CDR2 and/or CDR3 or at least part thereof (and inparticular at least CDR3 or at least part thereof). More preferably, anypart or fragment is such that it comprises at least one of the CDR's(and preferably at least CDR3 or part thereof) and at least one otherCDR (i.e., CDR1 or CDR2) or at least part thereof, preferably connectedby suitable framework sequence(s) or at least part thereof. Morepreferably, any part or fragment is such that it comprises at least oneof the CDR's (and preferably at least CDR3 or part thereof) and at leastpart of the two remaining CDR's, again preferably connected by suitableframework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect,such a part or fragment comprises at least CDR3, such as FR3, CDR3 andFR4 of the corresponding full length NB agent of the invention, i.e.,as, e.g., described in the International application WO 03/050531(Lasters et al.).

As already mentioned herein, it is also possible to combine two or moreof such parts or fragments (i.e., from the same or different NB agentsof the invention), i.e., to provide an analog and/or to provide furtherparts or fragments of a NB agent of the invention. It is, e.g., alsopossible to combine one or more parts or fragments of a NB agent of theinvention with one or more parts or fragments of a human V_(H) domain.

According to one aspect, the parts or fragments have a degree ofsequence identity of at least 50%, at least 60%, at least 70%, at least80%, and/or at least 90%, 95% or 99% or more with one of the NB agentsof SEQ ID NOs 1-22, 26-40, 87-88, and 102-103, and with SEQ ID NOs:96-99.

The parts and fragments, and nucleic acid sequences encoding the same,can be provided and optionally combined in any manner known per se. Forexample, such parts or fragments can be obtained by inserting a stopcodon in a nucleic acid that encodes a full-sized NB agent of theinvention, and then expressing the nucleic acid thus obtained in amanner known per se (e.g. as described herein). Alternatively, nucleicacids encoding such parts or fragments can be obtained by suitablyrestricting a nucleic acid that encodes a full-sized NB agent of theinvention or by synthesizing such a nucleic acid in a manner known perse. Parts or fragments may also be provided using techniques for peptidesynthesis known per se.

The invention in its broadest sense also comprises derivatives of the NBagents of the invention. Such derivatives can generally be obtained bymodification, and in particular by chemical and/or biological (e.g.enzymatic) modification, of the NB agents of the invention and/or of oneor more of the amino acid residues that form the NB agents of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the NB agent sequence that can be modified in such amanner (i.e., either on the polypeptide backbone but preferably on aside chain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

In one nonlimiting aspect, such a modification involves the introduction(e.g. by covalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the NB agent of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the NB agent of the invention. Example of suchfunctional groups will be clear to the skilled person.

In one nonlimiting aspect, such modification comprise the introduction(e.g. by covalent binding or in any other suitable manner) of one ormore functional groups that increase the half-life, the solubilityand/or the absorption of the NB agent of the invention, that reduce theimmunogenicity and/or the toxicity of the NB agent of the invention,that eliminate or attenuate any undesirable side effects of the NB agentof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the NB agents and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is, e.g., made to Remington's Pharmaceutical Sciences, 16thed., Mack Publishing Co., Easton, Pa. (1980). Such functional groupsmay, e.g., be linked directly (for example covalently) to a NB agent ofthe invention, or optionally via a suitable linker or spacer, as willagain be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethylene glycol) (PEG) or derivatives thereof (such asmethoxypoly(ethylene glycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); See, e.g., Chapman, Nat. Biotechnol., 54,531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54,453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003)and in WO 04/060965. Various reagents for pegylation of proteins arealso commercially available, e.g., from Nektar Therapeutics, USA.

In one embodiment, site-directed pegylation is used, in particular via acysteine-residue (see, e.g., Yang et al., Protein Engineering, 16, 10,761-770 (2003). In various embodiments, for this purpose, PEG isattached to a cysteine residue that naturally occurs in a NB agent ofthe invention. A NB agent of the invention may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a NB agentof the invention, all using techniques of protein engineering known perse to the skilled person.

In one embodiment, for the NB agents and proteins of the invention, aPEG is used with a molecular weight of more than 5000, such as more than10,000 and less than 200,000, such as less than 100,000, e.g., in therange of 20,000-80,000.

In one embodiment, modification comprises N-linked or O-linkedglycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the NB agent or polypeptide of the invention.

In one embodiment, modification may comprise the introduction of one ormore detectable labels or other signal-generating groups or moieties,depending on the intended use of the labeled NB agent. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and, e.g., include, but are not limited to, thefluorescent labels, phosphorescent labels, chemiluminescent labels,bioluminescent labels, radio-isotopes, metals, metal chelates, metalliccations, chromophores and enzymes, such as those mentioned on page 109of WO 08/020079. Other suitable labels will be clear to the skilledperson, and, e.g., include moieties that can be detected using NMR orESR spectroscopy.

Such labeled NB agents and polypeptides of the invention may, e.g., beused for in vitro, in vivo or in situ assays (including immunoassaysknown per se such as ELISA, RIA, EIA and other “sandwich assays”, etc.)as well as in vivo diagnostic and imaging purposes, depending on thechoice of the specific label.

In one embodiment, modification may involve the introduction of achelating group, e.g., to chelate one of the metals or metallic cationsreferred to above. Suitable chelating groups, e.g., include, withoutlimitation, diethyl-enetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

In one embodiment, modification may comprise the introduction of afunctional group that is one part of a specific binding pair, such asthe biotin-(strept)avidin binding pair. Such a functional group may beused to link the NB agent of the invention to another protein,polypeptide or chemical compound that is bound to the other half of thebinding pair, i.e., through formation of the binding pair. For example,a NB agent of the invention may be conjugated to biotin, and linked toanother protein, polypeptide, compound or carrier conjugated to avidinor streptavidin. For example, such a conjugated NB agent may be used asa reporter, e.g., in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may, e.g., also be used to bind the NB agent of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the inventive NB agent.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNB agents of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the NB agents of the invention may also be linked to a toxin or toa (cyto)toxic residue or moiety. Examples of toxic moieties, compoundsor residues which can be linked to a NB agent of the invention toprovide—, e.g.,—a cytotoxic compound will be clear to the skilled personand can, e.g., be found in the art references cited above and/or in thefurther description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatic modifications will be clear tothe skilled person and are within the scope of the invention. Suchmodifications may also be introduced for research purposes (e.g. tostudy function-activity relationships). Reference is, e.g., made toLundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

In one nonlimiting aspect, the derivatives are such that they bind to aDR5, e.g., human DR5, with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as provided hereinfor the NB agents of the invention.

As mentioned herein, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least one NBagent of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a NB agent of theinvention or corresponds to the amino acid sequence of a NB agent of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, e.g., 1-10 amino acid residues and preferably1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues,added at the amino terminal end, at the carboxy terminal end, or at boththe amino terminal end and the carboxy terminal end of the amino acidsequence of the NB agent.

Said amino acid residues may or may not change, alter or otherwiseappreciably influence the (biological) properties of the NB agent, andmay or may not add further functionality to the NB agent. For example,such amino acid residues:

a) can comprise an N-terminal Met residue, e.g., as result of expressionin a heterologous host cell or host organism; or

b) may form a signal sequence or leader sequence that directs secretionof the NB agent from a host cell upon synthesis. Suitable secretoryleader peptides will be clear to the skilled person, and may be asfurther described herein. Usually, such a leader sequence will be linkedto the N-terminus of the NB agent, although the invention in itsbroadest sense is not limited thereto; or

c) may form a sequence or signal that allows the NB agent to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows the NBagent to penetrate or cross a biological barrier such as a cellmembrane, a cell layer such as a layer of epithelial cells, a tumorincluding solid tumors, or the blood-brain-barrier. Examples of suchamino acid sequences will be clear to the skilled person and includethose mentioned in paragraph c) on page 112 of WO 08/020079; or

d) may form a “tag”, e.g., an amino acid sequence or residue that allowsor facilitates the purification of the NB agent, e.g., using affinitytechniques directed against said sequence or residue. Thereafter, saidsequence or residue may be removed (e.g. by chemical or enzymaticcleavage) to provide the NB agent sequence (for this purpose, the tagmay optionally be linked to the NB agent sequence via a cleavable linkersequence or contain a cleavable motif). Some non-limiting examples ofsuch residues are one or more of each of a cMyc tag (SEQ ID NO: 91),multiple histidine residues such as the His×6 tag (SEQ ID NO: 92), or acombination of a cMyc tag and the His×6 tag (SEQ ID NO: 93 or SEQ ID NO:94), PEGylation substrate tag (SEQ ID NO: 95), glutathione residues andother myc-tag (see, e.g., SEQ ID NO: 31 of WO 06/12282); or

e) may be one or more amino acid residues that have been functionalizedand/or that can serve as a site for attachment of functional groups.Suitable amino acid residues and functional groups will be clear to theskilled person and include, but are not limited to, the amino acidresidues and functional groups mentioned herein for the derivatives ofthe NB agents of the invention.

According to one aspect, a polypeptide of the invention comprises a NBagent of the invention that is fused at its amino terminal end, at itscarboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end, to at least one further amino acid sequence, i.e.,so as to provide a fusion protein comprising said NB agent of theinvention and the one or more further amino acid sequences. Such afusion is referred to herein as a “NB agent fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the NB agent, and may or may not add further functionality to the NBagent or the polypeptide of the invention. Preferably, the further aminoacid sequence is such that it confers one or more desired properties orfunctionalities to the NB agent or the polypeptide of the invention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the NB agent of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is, e.g., made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

In one nonlimiting aspect, such an amino acid sequence is an amino acidsequence that increases the half-life, the solubility, or theabsorption, reduces the immunogenicity or the toxicity, eliminates orattenuates undesirable side effects, and/or confers other advantageousproperties to and/or reduces the undesired properties of thepolypeptides of the invention, compared to the NB agent of the inventionper se. Some non-limiting examples of such amino acid sequences areserum proteins, such as human serum albumin (see, e.g., WO 00/27435) orhaptenic molecules (for example haptens that are recognized bycirculating antibodies, see, e.g., WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference ismade, e.g., to WO 00/27435 and WO 01/077137. According to the invention,a NB agent of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the NB agent of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference is,e.g., made to WO 07/112940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, e.g., human serum albumin or another serum protein such asIgG), so as to provide increased half-life in serum. Such amino acidsequences, e.g., include the NB agent described below, as well as thesmall peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, and to WO 2006/122787, WO2008/028977, WO 2008/043821, WO 2008/068280 and WO 2009/127691 by AblynxN.V.

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see, e.g., WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see again,e.g., WO 06/0122787); amino acid sequences that have or can provide anincreased half-life (see, e.g., WO 08/028977 by Ablynx N.V); amino acidsequences against human serum albumin that are cross-reactive with serumalbumin from at least one species of mammal, and in particular with atleast one species of primate (such as, without limitation, monkeys fromthe genus Macaca (such as, and in particular, cynomologus monkeys,a.k.a. “cyno” (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus), reference is again made to theU.S. provisional application 60/843,349); amino acid sequences that canbind to serum albumin in a pH independent manner (see, e.g., the U.S.provisional application 60/850,774 by Ablynx N.V. entitled “Amino acidsequences that bind to serum proteins in a manner that is essentiallyindependent of the pH, compounds comprising the same, and uses thereof”,filed on Oct. 11, 2006) and/or amino acid sequences that are conditionalbinders (see, e.g., the U.S. provisional application 60/850,775 byAblynx N.V. entitled “Amino acid sequences that bind to a desiredmolecule in a conditional manner”, filed on Oct. 11, 2006) and to WO2006/122787, WO 2008/028977, WO 2008/043821, WO 2008/068280 and WO2009/127691.

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional four-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a NB agent of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al.).

The at least one NB agent may be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a NB agent linked to a suitable C_(H)1 domaincould be used—together with suitable light chains—to generate antibodyfragments/structures analogous to conventional Fab fragments or F(ab′)₂fragments, but in which one or (in case of an F(ab′)₂ fragment) one orboth of the conventional V_(H) domains have been replaced by a NB agentof the invention. Also, two NB agents could be linked to a C_(H)3 domain(optionally via a linker) to provide a construct with increasedhalf-life in vivo.

In one aspect of the invention, one or more NB agents of the inventionare linked to one or more antibody parts, fragments or domains thatconfer one or more effector functions to the polypeptide of theinvention and/or may confer the ability to bind to one or more Fcreceptors. For example, for this purpose, and without being limitedthereto, the one or more further amino acid sequences may comprise oneor more C_(H)2 and/or C_(H)3 domains of an antibody, such as from aheavy chain antibody (as described herein) and more preferably from aconventional human 4-chain antibody; and/or may form (part of) and Fcregion, e.g., from IgG, from IgE or from another human Ig. For example,WO 94/04678 describes heavy chain antibodies comprising a Camelid V_(HH)domain or a humanized derivative thereof, in which the Camelidae C_(H)2and/or C_(H)3 domain have been replaced by human C_(H)2 and C_(H)3domains, so as to provide an immunoglobulin that consists of two heavychains each comprising a V_(HH) and human C_(H)2 and C_(H)3 domains (butno C_(H)1 domain), which immunoglobulin has the effector functionprovided by the C_(H)2 and C_(H)3 domains and which immunoglobulin canfunction without the presence of any light chains. Other amino acidsequences that can be suitably linked to the NB agents of the inventionso as to provide an effector function will be clear to the skilledperson, and may be chosen on the basis of the desired effectorfunction(s). Reference is, e.g., made to WO 04/058820, WO 99/42077 andWO 05/017148, as well as the review by Holliger and Hudson, supra.Coupling of a NB agent of the invention to an Fc portion may also leadto an increased half-life, compared to the corresponding NB agent of theinvention. For some applications, the use of an Fc portion and/or ofconstant domains (i.e., C_(H)2 and/or C_(H)3 domains) that conferincreased half-life without any biologically significant effectorfunction may also be suitable or even preferred. Other suitableconstructs comprising one or more NB agents and one or more constantdomains with increased half-life in vivo will be clear to the skilledperson, and may, e.g., comprise two NB agents linked to a C_(H)3 domain,optionally via a linker sequence. Generally, any fusion protein orderivatives with increased half-life will preferably have a molecularweight of more than 50 kD, the cut-off value for renal absorption.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the NB agent or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

In one aspect, a further amino acid sequence forms a sequence or signalthat allows the NB agent or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows the NBagent or polypeptide of the invention to penetrate or cross a biologicalbarrier such as a cell membrane, a cell layer such as a layer ofepithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and, e.g., include, but are not limitedto, those mentioned on page 118 of WO 08/020079. For some applications,in particular for those applications in which it is intended to kill acell that expresses the target against which the NB agents of theinvention are directed (e.g. in the treatment of cancer), or to reduceor slow the growth and/or proliferation of such a cell, the NB agents ofthe invention may also be linked to a (cyto)toxic protein orpolypeptide. Examples of such toxic proteins and polypeptides which canbe linked to a NB agent of the invention to provide—, e.g.,—a cytotoxicpolypeptide of the invention will be clear to the skilled person andcan, e.g., be found in the art references cited above and/or in thefurther description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

In one non-limiting aspect, said one or more further amino acidsequences comprise at least one further NB agent, so as to provide apolypeptide of the invention that comprises at least two, such as three,four, five or more NB agents, in which said NB agents may optionally belinked via one or more linker sequences. Polypeptides of the inventionthat comprise one, two or more V_(HH) constructs, e.g., as described onpages 119 and 120 of WO 08/020079, and at least one NB agent of theinvention, will also be referred to herein as “multivalent” polypeptidesof the invention, and the NB agent(s) present in such polypeptides willalso be referred to herein as being in a “multivalent format”.

Polypeptides of that contain at least two NB agents, in which at leastone NB agent is directed against a first antigen (i.e., against DR5,)and at least one NB agent is directed against a second antigen (i.e.,different from DR5), will also be referred to as “multispecific”polypeptides of the invention, and the NB agent present in suchpolypeptides will also be referred to herein as being in a“multispecific format”. Thus, e.g., a “bispecific” polypeptide of theinvention is a polypeptide that comprises at least one NB agent directedagainst a first antigen (i.e., DR5), more preferably three, four, fiveor more NB agents directed against DR5 and at least one further NB agentdirected against a second antigen (i.e., different from DR5), whereas a“trispecific” polypeptide of the invention is a polypeptide thatcomprises at least one NB agent directed against a first antigen (i.e.,one epitope of DR5), at least one further NB agent directed against asecond antigen (i.e., a different epitope from DR5 or an antigendifferent from TRAIIL-receptor,) and at least one further NB agentdirected against a third antigen (i.e., different from both DR5 and thesecond antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention, comprising a firstNB agent directed against DR5, and a second NB agent directed against asecond antigen, in which said first and second NB agent may optionallybe linked via a linker sequence; whereas a trispecific polypeptide ofthe invention in its simplest form is a trivalent polypeptide of theinvention, comprising a first NB agent directed against DR5, a second NBagent directed against a second antigen and a third NB agent directedagainst a third antigen, in which said first, second and third NB agentmay optionally be linked via one or more, and in particular one andmore, in particular two, linker sequences.

As will be clear from the description herein, the invention is notlimited the above, in the sense that a multispecific polypeptide of theinvention may comprise at least one or more NB agents against DR5, andany number of NB agents directed against one or more antigens differentfrom DR5.

When reference is made to a specific multivalent or multispecificpolypeptide of the invention, it should be noted that this encompassesany order or arrangements of the relevant NB agents, unless explicitlyindicated otherwise.

Furthermore, it is within the scope of the invention that thepolypeptides of the invention contain two or more NB agents and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to,e.g., WO 96/34103 and WO 99/23221. Some other examples of some specificmultispecific and/or multivalent polypeptide of the invention can befound in the applications by Ablynx N.V. referred to herein.

One non-limiting example of a multispecific polypeptide of the inventioncomprises at least one NB agent of the invention and at least one NBagent that provides for an increased half-life. Such NB agents may,e.g., be NB agents that are directed against a serum protein, and inparticular a human serum protein, such as human serum albumin,thyroxine-binding protein, (human) transferrin, fibrinogen, animmunoglobulin such as IgG, IgE or IgM, or against one of the serumproteins listed in WO 04/003019. Of these, NB agents that can bind toserum albumin (and in particular human serum albumin) or to IgG (and inparticular human IgG, see, e.g., NANOBODY™ VH-1 described in the reviewby Muyldermans, supra) are particularly preferred (although, e.g., forexperiments in mice or primates, NB agents against or cross-reactivewith mouse serum albumin (MSA) or serum albumin from said primate,respectively, can be used. However, for pharmaceutical use, NB agentsagainst human serum albumin or human IgG will usually be preferred). NBagents that provide for increased half-life and that can be used in thepolypeptides of the invention include the NB agents directed againstserum albumin that are described in WO 04/041865, in WO 06/122787 and inthe further patent applications by Ablynx N.V., such as those mentionedherein.

For example, the some preferred NB agents that provide for increasedhalf-life for use in the present invention include NANOBODIES™ that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see, e.g., WO 06/0122787);NANOBODIES™ that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see, e.g.,WO 06/0122787); NANOBODIES™ that have or can provide an increasedhalf-life (see, e.g., the U.S. provisional application 60/843,349 byAblynx N.V mentioned herein); NANOBODIES™ against human serum albuminthat are cross-reactive with serum albumin from at least one species ofmammal, and in particular with at least one species of primate (such as,without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomologus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus)) (see, e.g., theU.S. provisional application 60/843,349 by Ablynx N.V); NANOBODIES™ thatcan bind to serum albumin in a pH independent manner (see, e.g., theU.S. provisional application 60/850,774 by Ablynx N.V. mentioned herein)and/or NANOBODIES™ that are conditional binders (see, e.g., the U.S.provisional application 60/850,775 by Ablynx N.V.).

Some particularly preferred NANOBODIES™ that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the NANOBODIES™ ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III therein) of which ALB-8 (SEQ ID NO: 62 in WO06/122787) is particularly preferred.

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or more NB agentsof the invention, at least one NB agent against human serum albumin.

In one embodiment, any polypeptides of the invention with increasedhalf-life that contain one or more NB agents of the invention, and anyderivatives of NB agents of the invention or of such polypeptides thathave an increased half-life, have a half-life that is at least 1.5times, and/or at least 2 times, and/or at least 5 times, e.g., at least10 times or more than 20 times, greater than the half-life of thecorresponding NB agent of the invention per se. For example, such aderivative or polypeptides with increased half-life may have a half-lifethat is increased with more than 1 hours, and/or more than 2 hours,and/or more than 6 hours, such as more than 12 hours, or even more than24, 48 or 72 hours, compared to the corresponding NB agent of theinvention per se.

In one non-limiting aspect of the invention, such derivatives orpolypeptides exhibit a serum half-life in human of at least about 12hours, and/or at least 24 hours, and/or at least 48 hours, and/or atleast 72 hours or more. For example, such derivatives or polypeptidesmay have a half-life of at least 5 days (such as about 5 to 10 days),and/or at least 9 days (such as about 9 to 14 days), and/or at leastabout 10 days (such as about 10 to 15 days), and/or at least about 11days (such as about 11 to 16 days), and/or at least about 12 days (suchas about 12 to 18 days or more), and/or more than 14 days (such as about14 to 19 days).

According to one aspect of the invention, such polypeptides are capableof binding to one or more molecules that can increase the half-life ofthe polypeptide in vivo.

Such polypeptides of the invention are stabilized in vivo and theirhalf-life increased by binding to molecules that resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

Another non-limiting example of a multispecific polypeptide of theinvention comprises at least one NB agent of the invention and at leastone NB agent that directs the polypeptide of the invention towards,and/or that allows the polypeptide of the invention to penetrate or toenter into specific organs, tissues, cells, or parts or compartments ofcells, and/or that allows the NB agent to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Examples of such NB agents include NB agents thatare directed towards specific cell-surface proteins, markers or epitopesof the desired organ, tissue or cell (for example cell-surface markersassociated with tumor cells), and the single-domain brain targetingantibody fragments described in WO 02/057445 and WO 06/040153, of whichFC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO06/040154) are preferred examples.

Linkers

In the polypeptides or compounds of the invention, the one or morebinding polypeptides against DR5, such as NB agents and the one or morepolypeptides may be directly linked to each other (as, e.g., describedin WO 99/23221) and/or may be linked to each other via one or moresuitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as, e.g., linkers that are used in the art to constructdiabodies or ScFv fragments (in this respect, however, its should benoted that, whereas in diabodies and in ScFv fragments, the linkersequence used should have a length, a degree of flexibility and otherproperties that allow the pertinent V_(H) and V_(L) domains to cometogether to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each NB agent by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, e.g., between 5 and50, preferably between 1 and 35, such as between 1 and 10 amino acidresidues. Some preferred examples of such amino acid sequences includegly-ser linkers, e.g., of the type (gly_(x)ser_(y))_(z), such as (forexample (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and theGS30, GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see, e.g., WO 06/040153 and WO 06/122825), as well ashinge-like regions, such as the hinge regions of naturally occurringheavy chain antibodies or similar sequences (e.g., such as described inWO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.Nonlimiting examples include, e.g., poly(ethylene glycol) moieties,e.g., such as those used to link antibody domains. See, e.g., WO04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for DR5, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

In one embodiment, in multivalent polypeptides of the invention thatcomprise, e.g., NB agents directed against a multimeric antigen (such asa multimeric receptor or other protein), the length and flexibility ofthe linker may be such that it allows each NB agent of the inventionpresent in the polypeptide to bind to the antigenic determinant on eachof the subunits of the multimer. Similarly, in a multispecificpolypeptide of the invention that comprises NB agents directed againsttwo or more different antigenic determinants on the same antigen (forexample against different epitopes of an antigen and/or againstdifferent subunits of a multimeric receptor, channel or protein), thelength and flexibility of the linker may be such that it allows each NBagent to bind to its intended antigenic determinant. Again, based on thedisclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is within the scope of the invention that the linker(s) used conferone or more other favorable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the NB agents of theinvention). For example, linkers containing one or more charged aminoacid residues can provide improved hydrophilic properties, whereaslinkers that form or contain small epitopes or tags can be used for thepurposes of detection, identification and/or purification. Again, basedon the disclosure herein, the skilled person will be able to determinethe optimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thereto. For example, when a polypeptideof the invention comprises three or more NB agents, it is possible tolink them by use of a linker with three or more “arms”, with each “arm”being linked to a NB agent, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention further comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNB agents of the invention, i.e., as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedhereinabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated form, as defined herein.

Method of Manufacturing the NB Agents of the Invention

The amino acid sequences, NB agents, polypeptides, compounds and nucleicacids of the invention can be prepared in a manner known per se, as willbe clear to the skilled person from the further description herein. Forexample, the NB agents and polypeptides of the invention can be preparedin any manner known per se for the preparation of antibodies and inparticular for the preparation of antibody fragments (including but notlimited to (single) domain antibodies and ScFv fragments). Somepreferred, but non-limiting methods for preparing the amino acidsequences, NB agents, polypeptides and nucleic acids include the methodsand techniques described herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, NB agent and/or a polypeptide ofthe invention generally comprises the steps of:

-   -   a) the expression, in a suitable host cell or host organism        (also referred to herein as a “host of the invention”) or in        another suitable expression system of a nucleic acid that        encodes said amino acid sequence, NB agent or polypeptide of the        invention (also referred to herein as a “nucleic acid of the        invention”), optionally followed by:    -   b) isolating and/or purifying the amino acid sequence, NB agent        or polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   -   a) cultivating and/or maintaining a host of the invention under        conditions that are such that said host of the invention        expresses and/or produces at least one amino acid sequence, NB        agent and/or polypeptide of the invention;        optionally followed by:    -   b) isolating and/or purifying the amino acid sequence, NB agent        or polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA. In one embodiment, a polynucleotide NB agent is inthe form of double stranded DNA. For example, the nucleotide NBsequences of the invention may be genomic DNA, cDNA or synthetic DNA(such as DNA with a codon usage that has been specifically adapted forexpression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated form, as defined herein.

The nucleic acid of the invention may be in the form of, be present inand/or be part of a vector, such as, e.g., a plasmid, cosmid or YAC,which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can,e.g., be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. As will be clear tothe skilled person, to prepare a nucleic acid of the invention,including several nucleotide sequences, e.g., at least one nucleotidesequence encoding a NB agent and, e.g., nucleic acids encoding one ormore linkers, such sequences can be linked together in a suitablemanner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using, e.g., a sequence of a naturallyoccurring form of DR5 as a template. These and other techniques will beclear to the skilled person, and reference is again made to the standardhandbooks, such as Sambrook et al. and Ausubel et al., mentioned herein,as well as the Examples below.

The nucleic acid of the invention may be in the form of, be present inand/or be part of a genetic construct, as will be clear to the personskilled in the art and, e.g., as described on pages 131-134 of WO08/020079. Such genetic constructs generally comprise at least onenucleic acid of the invention that is optionally linked to one or moreelements of genetic constructs known per se, such as, e.g., one or moresuitable regulatory elements (such as a suitable promoter(s),enhancer(s), terminator(s), etc.) and the further elements of geneticconstructs referred to herein. Such genetic constructs comprising atleast one nucleic acid of the invention will also be referred to hereinas “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA. The geneticconstructs of the invention may be in a form suitable for transformationof the intended host cell or host organism, in a form suitable forintegration into the genomic DNA of the intended host cell or in a formsuitable for independent replication, maintenance and/or inheritance inthe intended host organism. For instance, the genetic constructs of theinvention may be in the form of a vector, such as, e.g., a plasmid,cosmid, YAC, a viral vector or transposon. In particular, the vector maybe an expression vector, i.e., a vector that can provide for expressionin vitro and/or in vivo (e.g. in a suitable host cell, host organismand/or expression system).

The nucleic acid of the invention may be in the form of, be present inand/or be part of a genetic construct. Such genetic constructs generallycomprise at least one nucleic acid of the invention that is optionallylinked to one or more elements of genetic constructs known per se, suchas, e.g., one or more suitable regulatory elements (such as a suitablepromoter(s), enhancer(s), terminator(s), etc.) and the further elementsof genetic constructs referred to herein. Other suitable geneticconstructs known in the art are contemplated as being within the scopeof the invention, including e.g., those described on pages 131-134 of WO08/020079. Such genetic constructs comprising at least one nucleic acidof the invention will also be referred to herein as “genetic constructsof the invention”.

In one non-limiting embodiment, a genetic construct of the inventioncomprises a) at least one nucleic acid of the invention; wherein saidpolynucleotide NB agent is operably connected to b) one or moreregulatory elements, such as a promoter and optionally a suitableterminator; and optionally c) one or more further elements of geneticconstructs known per se.

In one embodiment, in the genetic constructs of the invention, said atleast one nucleic acid of the invention and said regulatory elements,and optionally said one or more further elements, are “operably linked”to each other, by which is generally meant that they are in a functionalrelationship with each other. For instance, a promoter is considered“operably linked” to a coding sequence if said promoter is able toinitiate or otherwise control/regulate the transcription and/or theexpression of a coding sequence (in which said coding sequence should beunderstood as being “under the control of said promotor). Generally,when two nucleotide sequences are operably linked, they will be in thesame orientation and usually also in the same reading frame. They willusually be essentially contiguous, although this may not be required.The phrases “operably connected” and “operably linked” are used in thealternative.

The terms “regulatory element”, “promoter”, “terminator” and “operablyconnected” have their usual meaning in the art. The “further elements”present in the genetic constructs may be, e.g., a 3′- or 5′-UTRsequences, leader sequences, selection markers, expressionmarkers/reporter genes, and/or elements that may facilitate or increase(the efficiency of) transformation or integration. These and othersuitable elements for such genetic constructs will be clear to theskilled person, and may for instance depend upon the type of constructused, the intended host cell or host organism; the manner in which thenucleotide sequences of the invention of interest are to be expressed(e.g. via constitutive, transient or inducible expression); and/or thetransformation technique to be used. For example, regulatory sequences,promoters and terminators known per se for the expression and productionof antibodies and antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments) may be used in anessentially analogous manner.

In one embodiment, the regulatory and further elements of the geneticconstructs of the invention are such that they are capable of providingtheir intended biological function in the intended host cell or hostorganism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that (forexample) said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked (as defined herein). Some particularly preferred promotersinclude, but are not limited to, promoters known per se for theexpression in the host cells mentioned herein; and in particularpromoters for the expression in the bacterial cells.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.,for expression and/or production of the amino acid sequence, NB agent orpolypeptide of the invention. Suitable hosts or host cells will be clearto the skilled person, and may, e.g., be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism, e.g., those described on pages 134and 135 of WO 08/020079; as well as all other hosts or host cells knownper se for the expression and production of antibodies and antibodyfragments (including but not limited to (single) domain antibodies andScFv fragments), which will be clear to the skilled person. Reference isalso made to the general background art cited hereinabove, as well asto, e.g., WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al., (1998),supra; Riechmann and Muyldermans, (1999), supra; van der Linden, (2000),supra; Thomassen et al., (2002), supra; Joosten et al., (2003), supra;Joosten et al., (2005), supra; and the further references cited herein.

The NB amino acid sequences and polypeptides of the invention can beintroduced and expressed in one or more cells, tissues or organs of amulticellular organism, e.g., for prophylactic and/or therapeuticpurposes (e.g., as a gene therapy), as further described on pages 135and 136 of in WO 08/020079 and in the further references cited in WO08/020079.

For expression of the NB agents in a cell, they may be expressed asso-called “intrabodies”, as, e.g., described in WO 94/02610, WO 95/22618and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S.(1997) Intracellular Antibodies: Development and Applications. Landesand Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170.

The amino NB acid sequences and polypeptides of the invention can, e.g.,also be produced in the milk of transgenic mammals, e.g., in the milk ofrabbits, cows, goats or sheep (see, e.g., U.S. Pat. No. 6,741,957, U.S.Pat. No. 6,304,489 and U.S. Pat. No. 6,849,992 for general techniquesfor introducing transgenes into mammals), in plants or parts of plantsincluding but not limited to their leaves, flowers, fruits, seed, rootsor tubers (for example in tobacco, maize, soybean or alfalfa) or in,e.g., pupae of the silkworm Bombix mori.

Furthermore, the NB amino acid sequences and polypeptides of theinvention can be expressed and/or produced in cell-free expressionsystems, and suitable examples of such systems will be clear to theskilled person. Some non-limiting examples include expression in thewheat germ system; in rabbit reticulocyte lysates; or in the E. coliZubay system.

As mentioned herein, one of the advantages of the use of NB agentsexpressed from plasmids or vectors is that the polypeptides basedthereon can be prepared through expression in a suitable bacterialsystem, and suitable bacterial expression systems, vectors, host cells,regulatory elements, etc., will be clear to the skilled person, e.g.,from the references cited above. It should however be noted that theinvention in its broadest sense is not limited to expression inbacterial systems.

In one embodiment, codon usage for a polynucleotide sequence of theinvention is optimization by referring to the frequency of codon usageof the particular host cell/host organism. The resulting nucleotidesequence is a “codon optimized” NB nucleotide sequence. Codon optimizedNB sequences may be made for expression in prokaryotic or eukaryotichost cells/host organisms. Particular examples include the expressionsystems provided herein.

In one embodiment of the invention an expression system (either in vivoor in vitro), e.g., such as a bacterial expression system, is used thatprovides the polypeptides of the invention in a form that is suitablefor pharmaceutical use, and such expression systems will again be clearto the skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of NB agents or NB agent-containing proteintherapeutics include strains of E. coli, Pichia pastoris, S. cerevisiaethat are suitable for large scale expression/production/fermentation,and in particular for large scale pharmaceutical (i.e., GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned herein.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a NB agent-containingrecombinant protein for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e., thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression. Preferably, either a humancell or cell line is used (i.e., leading to a protein that essentiallyhas a human glycosylation pattern) or another mammalian cell line isused that can provide a glycosylation pattern that is essentially and/orfunctionally the same as human glycosylation or at least mimics humanglycosylation. Generally, prokaryotic hosts such as E. coli do not havethe ability to glycosylate proteins, and the use of lower eukaryotessuch as yeast usually leads to a glycosylation pattern that differs fromhuman glycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, NB agent orpolypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, NB agent or polypeptide of the invention is glycosylated.According to another non-limiting aspect of the invention, the aminoacid sequence, NB agent or polypeptide of the invention isnon-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, NB agent or polypeptide of the invention isproduced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned herein.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, NB agent or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned herein.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, NB agent or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acidsequences, NB agents and the polypeptides of the invention, the NB aminoacid sequences and polypeptides of the invention can be produced eitherintracellullarly (e.g. in the cytosol, in the periplasma or in inclusionbodies) and then isolated from the host cells and optionally furtherpurified; or can be produced extracellularly (e.g. in the medium inwhich the host cells are cultured) and then isolated from the culturemedium and optionally further purified. Thus, according to onenon-limiting aspect of the invention, the amino acid sequence, NB agentor polypeptide of the invention is an amino acid sequence, NB agent orpolypeptide that has been produced intracellularly and that has beenisolated from the host cell, and in particular from a bacterial cell orfrom an inclusion body in a bacterial cell. According to anothernon-limiting aspect of the invention, the amino acid sequence, NB agentor polypeptide of the invention is an amino acid sequence, NB agent orpolypeptide that has been produced extracellularly, and that has beenisolated from the medium in which the host cell is cultivated.

A more complete discussion of expression systems is provided, e.g., onpages 138 and 139 of WO 08/020079. Included are a non-limiting list ofpromoters for use with these host cells, e.g., including those mentionedon pages 139 and 140 of WO 08/020079. Also included are a non-limitinglist of secretory sequences for use with these host cells, e.g.,including those mentioned on page 140 of WO 08/020079.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct used.Reference is again made to the handbooks and patent applicationsmentioned herein.

It will also be clear to the skilled person that the amino acidsequence, NB agent or polypeptide of the invention may (first) begenerated in an immature form (as mentioned herein), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, NB agent orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, NB agent or polypeptide of the invention maythen be isolated from the host cell/host organism and/or from the mediumin which said host cell or host organism is cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, NB agent or polypeptide of the invention) and/or preparativeimmunological techniques (i.e., using antibodies against the amino acidsequence to be isolated).

Pharmaceutical Preparation or Compositions

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or composition comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein. Such a pharmaceutical preparation orcomposition will generally be referred to herein as a “pharmaceuticalcomposition”. A pharmaceutical preparation or composition for use in anon-human organism will generally be referred to herein as a “veterinarycomposition”.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one NB agent of the invention or at least one polypeptide of theinvention and at least one suitable carrier, diluent or excipient (i.e.,suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the NB amino acid sequences and polypeptides of the inventioncan be formulated and administered in any suitable manner known per se.Reference is, e.g., made to the general background art cited above (andin particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867and WO 08/020079) as well as to the standard handbooks, such asRemington's Pharmaceutical Sciences, 18^(th) Ed., Mack PublishingCompany, USA (1990), Remington, the Science and Practice of Pharmacy,21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook ofTherapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see,e.g., pages 252-255).

The NB amino acid sequences and polypeptides of the invention may beformulated and administered in any manner known per se for conventionalantibodies and antibody fragments (including ScFv's and diabodies) andother pharmaceutically active proteins. Such formulations and methodsfor preparing the same will be clear to the skilled person, and, e.g.,include preparations suitable for parenteral administration (for exampleintravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal,intra-arterial or intrathecal administration) or for topical (i.e.,transdermal or intradermal) administration.

Preparations for parenteral administration may, e.g., be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations, e.g., include, without limitation, those mentioned on page143 of WO 08/020079. In one embodiment, the preparation is an aqueoussolution or suspension.

The NB amino acid sequences and polypeptides of the invention can beadministered using gene therapy methods of delivery. See, e.g., U.S.Pat. No. 5,399,346, which is incorporated by reference for its genetherapy delivery methods. Using a gene therapy method of delivery,primary cells transfected with the gene encoding an amino acid sequence,NB agent or polypeptide of the invention can additionally be transfectedwith tissue specific promoters to target specific organs, tissue,grafts, tumors, or cells and can additionally be transfected with signaland stabilization sequences for subcellularly localized expression.

Thus, the NB amino acid sequences and polypeptides of the invention maybe systemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the NB amino acid sequences and polypeptides of theinvention may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of the amino acid sequence, NBagent or polypeptide of the invention. Their percentage in thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of the amino acid sequence, NB agent orpolypeptide of the invention in such therapeutically useful compositionsis such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavoring agents, e.g., those mentioned on pages 143-144 of WO08/020079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the NB amino acid sequences and polypeptides of theinvention, sucrose or fructose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the NB amino acid sequences andpolypeptides of the invention may be incorporated into sustained-releasepreparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The NB amino acid sequences and polypeptides of the invention may alsobe administered intravenously or intraperitoneally by infusion orinjection. Particular examples are as further described on pages 144 and145 of WO 08/020079.

For topical administration, the NB amino acid sequences and polypeptidesof the invention may be applied in pure form, i.e., when they areliquids. However, it will generally be desirable to administer them tothe skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.Particular examples are as further described on page 145 of WO08/020079.

Generally, the concentration of the NB amino acid sequences andpolypeptides of the invention in a liquid composition, such as a lotion,will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. Theconcentration in a semi-solid or solid composition such as a gel or apowder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the NB amino acid sequences and polypeptides of theinvention required for use in treatment will vary not only with theparticular amino acid sequence, NB agent or polypeptide selected butalso with the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician. Also thedosage of the NB amino acid sequences and polypeptides of the inventionvaries depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, e.g., as two,three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one diseases and disorders associated withDR5, said method comprising administering, to a subject in need thereof,a pharmaceutically active amount of an amino acid sequence of theinvention, of a NB agent of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with DR5, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signaling in which DR5 is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a NB agent of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In one embodiment, the inventionrelates to a method for the prevention and/or treatment of at least onedisease or disorder that can be treated by modulating DR5, itsbiological or pharmacological activity, and/or the biological pathwaysor signaling in which DR5 are involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a NB agent of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In one embodiment, saidpharmaceutically effective amount may be an amount that is sufficient tomodulate DR5, its biological or pharmacological activity, and/or thebiological pathways or signaling in which DR5 is involved; and/or anamount that provides a level of the amino acid sequence of theinvention, of a NB agent of the invention, of a polypeptide of theinvention in the circulation that is sufficient to modulate DR5, itsbiological or pharmacological activity, and/or the biological pathwaysor signaling in which DR5 is involved.

In one embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by administering a NB amino acid sequence orpolypeptide of the invention, or a NB nucleotide construct of theinvention encoding the same, and/or of a pharmaceutical compositioncomprising the same, to a patient. In one embodiment, the methodcomprises administering a pharmaceutically active amount of a NB aminoacid sequence or polypeptide of the invention, or a NB nucleotideconstruct of the invention encoding the same, and/or of a pharmaceuticalcomposition comprising the same to a subject in need thereof.

In one embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by enhancing cell apoptosis in specific cellsor in a specific tissue of a subject to be treated (and in particular,by enhancing cell apoptosis in cancer cells or in a tumor present in thesubject to be treated), said method comprising administering apharmaceutically active amount of a NB amino acid sequence orpolypeptide of the invention, or a NB nucleotide construct of theinvention encoding the same, and/or of a pharmaceutical compositioncomprising the same, to a subject in need thereof.

In one embodiment, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, a NBamino acid sequence or polypeptide of the invention, or a NB nucleotideconstruct of the invention encoding the same, and/or of a pharmaceuticalcomposition comprising the same.

In one embodiment, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of a NBamino acid sequence or polypeptide of the invention, or a NB nucleotideconstruct of the invention encoding the same, and/or of a pharmaceuticalcomposition comprising the same.

In the above methods, the amino acid sequences, NB agents and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, NB agents and/or polypeptides of the inventionand/or the compositions comprising the same can, e.g., be administeredorally, intraperitoneally (e.g. intravenously, subcutaneously,intramuscularly, or via any other route of administration thatcircumvents the gastrointestinal tract), intranasally, transdermally,topically, by means of a suppository, by inhalation, again depending onthe specific pharmaceutical formulation or composition to be used. Theclinician will be able to select a suitable route of administration anda suitable pharmaceutical formulation or composition to be used in suchadministration, depending on the disease or disorder to be prevented ortreated and other factors well known to the clinician.

The amino acid sequences, NB agents and/or polypeptides of the inventionand/or the compositions comprising the same are administered accordingto a regime of treatment that is suitable for preventing and/or treatingthe disease or disorder to be prevented or treated. The clinician willgenerally be able to determine a suitable treatment regimen, dependingon factors such as the disease or disorder to be prevented or treated,the severity of the disease to be treated and/or the severity of thesymptoms thereof, the specific amino acid sequence, NB agent orpolypeptide of the invention to be used, the specific route ofadministration and pharmaceutical formulation or composition to be used,the age, gender, weight, diet, general condition of the patient, andsimilar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, NB agents and/or polypeptides of theinvention, or of one or more compositions comprising the same, in one ormore pharmaceutically effective amounts or doses. The specific amount(s)or doses to administered can be determined by the clinician, again basedon the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,NB agent and polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the NB amino acid sequences and polypeptides of theinvention will generally be administered in an amount between 1 gram and0.01 microgram per kg body weight per day, preferably between 0.1 gramand 0.1 microgram per kg body weight per day, such as about 1, 10, 100or 1000 microgram per kg body weight per day, either continuously (e.g.by infusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, e.g., on the basis of the factors cited above and hisexpert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

In one embodiment, a single contiguous amino acid sequence, NB agent orpolypeptide of the invention will be used, whether the translatedsequences contains a single domain or multiple monovalent or multivalentdomains. In one embodiment two or more amino acid sequences, NB agentsand/or polypeptides of the invention are provided in combination.

The NB agents, amino acid sequences and polypeptides of the inventionmay be used in combination with one or more further pharmaceuticallyactive compounds or principles, i.e., as a combined treatment regimen,which may or may not lead to a synergistic effect. Again, the clinicianwill be able to select such further compounds or principles, as well asa suitable combined treatment regimen, based on the factors cited aboveand his expert judgment.

In particular, the NB amino acid sequences and polypeptides of theinvention may be used in combination with other pharmaceutically activecompounds or principles that are or can be used for the preventionand/or treatment of the diseases and disorders cited herein, as a resultof which a synergistic effect may or may not be obtained. Examples ofsuch compounds and principles, as well as routes, methods andpharmaceutical formulations or compositions for administering them willbe clear to the clinician, and generally include the cytostatic activeprinciples usually applied for the treatment of the tumor to be treated.

Specific contemplated combinations for use with the NB agents of theinvention include, but are not limited to, e.g., taxol; gemcitobine;cisplatin; cIAP inhibitors (such as inhibitors to cIAP1, cIAP2 and/orXIAP); MEK inhibitors including but not limited to, e.g., U0126,PD0325901; bRaf inhibitors including but not limited to, e.g., RAF265;and mTOR inhibitors including but not limited to, e.g., RAD001. Specificcombinations are provided herein and in the Examples.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may, e.g., be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, NB agent or polypeptide of the invention in the preparation ofa pharmaceutical composition for prevention and/or treatment of at leastone diseases and disorders associated with DR5; and/or for use in one ormore of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. In veterinaryapplications, the subject to be treated includes any animal raised forcommercial purposes or kept as a pet. As will be clear to the skilledperson, the subject to be treated will in particular be a personsuffering from, or at risk of, the diseases and disorders mentionedherein.

The invention relates to the use of a NB amino acid sequence orpolypeptide of the invention, or a NB nucleotide encoding the same, inthe preparation of a pharmaceutical composition for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by administering a NB amino acid sequence orpolypeptide of the invention, or a NB nucleotide encoding the same,and/or a pharmaceutical composition of the same to a patient.

More in particular, the invention relates to the use of a NB amino acidsequence or polypeptide of the invention, or a NB nucleotide encodingthe same, in the preparation of a pharmaceutical composition for theprevention and/or treatment of diseases and disorders associated withDR5, and in particular for the prevention and treatment of one or moreof the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more NB aminoacid sequence or polypeptide of the invention, or NB nucleotide encodingthe same, and/or a pharmaceutical composition of the same, may also besuitably combined with one or more other active principles, such asthose mentioned herein.

In one embodiment, although the use of the exemplary NB agents of theinvention is much preferred, it will be clear that on the basis of thedescription herein, the skilled person will be able to design and/orgenerate, in an analogous manner, other amino acid sequences and inparticular (single) domain antibodies against DR5, as well aspolypeptides comprising such (single) domain antibodies, and/ornucleotides that encode them.

For example, it will be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned herein for the NBagents of the invention onto such (single) domain antibodies or otherprotein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see, e.g., those mentioned in WO 08/020079. For example,techniques known per se for grafting mouse or rat CDR's onto humanframeworks and scaffolds can be used in an analogous manner to providechimeric proteins comprising one or more of the CDR's of the NB agentsof the invention and one or more human framework regions or sequences.

It should also be noted that, when the NB agents of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned herein, these CDR sequences can be obtained in any mannerknown per se, e.g., using one or more of the techniques described in WO08/020079.

Further uses of the amino acid sequences, NB agent s, polypeptides,nucleic acids, genetic constructs and hosts and host cells of theinvention will be clear to the skilled person based on the disclosureherein. For example, and without limitation, the amino acid sequences ofthe invention can be linked to a suitable carrier or solid support so asto provide a medium than can be used in a manner known per se to purifyDR5 from compositions and preparations comprising the same. Derivativesof the amino acid sequences of the invention that comprise a suitabledetectable label can also be used as markers to determine (qualitativelyor quantitatively) the presence of DR5 in a composition or preparationor as a marker to selectively detect the presence of DR5 on the surfaceof a cell or tissue (for example, in combination with suitable cellsorting techniques).

The invention will now be further described by means of the followingnon-limiting experimental part.

1. Example I 1.1. Human and Cyno DR5 Cloning and Protein Preparation

1.1.1. Cloning of Human Long Form DR5 Extracellular Domain (ECD) andCyno Long and Short Form DR5 ECD

The human long form DR5 ECD (aa55-213) is cloned by RT-PCR. Total RNAare isolated from Jurkat and Raji cells by Qiagen's RNeasy mini Kit (CatNo. 74104). cDNA is made by the SuperScript II First Strand SynthesisSystem (Invitrogen, Cat: 11904-018) then are amplified by High FidelityPlatinum Taq DNA Polymerase (Invitrogen, Cat no. 11304-011) usingstandard protocol: 94° C. for 2 min followed by 30 cycles of 95° C./30seconds, 55° C./30 seconds, 72° C./60 seconds and a final incubation at72° C. for 7 min. The forward and reverse primers used for PCR are:5′-CTGATCACCC AACAAGACCT AG-3′ (SEQ ID NO: 83) and 5′-GCCTGAGAGAGAACAGGGAG A-3′ (SEQ ID NO: 84) respectively. The resulting 476 byfragment is then ligated into E. coli expression vector pBAD/Thio-TOPO(Invitrogen, Cat No. K370-01). Positive clones are identified by PCR andconfirmed by DNA sequencing.

The full length cyno DR5 gene is originally cloned from cynomolgus livercDNA (BioChain Institute, Inc. USA) by PR-PCR. The forward(5′-CACCATGGAA CAACGGGGAC AGAACGCC-3′) (SEQ ID NO: 85) and reverse(5′-TTAGGACATG GCAGAGTCTG CATTACCTTC-3′) (SEQ ID NO: 86) primers aredesigned based on the human DR5 nucleotide sequence, including the start(ATG) and stop (TAA) codons respectively. High Fidelity Platinum Taq DNAPolymerase (Invitrogen, Cat no. 11304-011) is used for PCR and theresulting fragments are ligated into the pcDNA3.1 directional TOPOvector. The positive colonies are identified by DNA sequencing. Similarto human DR5 gene, two splicing alternatives of cyno DR5 gene areidentified. One has the long form ECD, and the other has the short formECD.

Both cyno long (aa55-213) and short (aa55-174) form of DR5 ECD arefurther cloned into pBAD/Thio-TOPO vector (Invitrogen, Cat No. K370-01).Positive clones are identified by PCR and are confirmed by DNAsequencing.

1.1.2. Establishment of CHO Cell Lines Expressing Cell Surface Cyno DR5

Chinese Hamster Ovary cells (CHO-K1) are transfected at 90-95%confluency using FuGENE6 (Roche Applied Sciences) and 1 μgpcDNA3.1-cynoDR5 expression vector. Transfected cells are selected by500 μg/ml Geneticin (G418) and are subcloned by cell sorting.

1.1.3. Expression and Purification of Thioredoxin-DR5ECD-his6 Fusionfrom E. coli

For expression of DR5 ECD, pBAD/Thio-DR5ECD is transformed into E. colistrain TOP10 (Invitrogen, Cat No. C4040-03). A single colony is used toinoculate 100 ml LB containing 100 μg/ml ampicillin. This culture isincubated overnight at 37° C. with shaking at 225 rpm. Three liters ofLB/ampicillin are inoculated with 30 ml of the overnight culture andshaken at 37° C. till the OD₆₀₀ reaches 0.5. The culture is then inducedwith 0.02% arabinose for 3 hours at 37° C. The bacterial pellet isresuspended in 21 ml lysis buffer (PBS with 0.3M NaCl, 0.4% Triton X-100and 10 mM imidazole) containing protease inhibitors (Roche, Cat No.1836153). Lysate is sonicated two times for 60 seconds followed bycentrifugation at 20,000×g for 20 min. Soluble His×6 tagged DR5 ECD ispurified by nickel chelate affinity chromatography. Soluble His×6 taggedDR5 ECD is created by inserting the sequence encoding the polypeptidetag of SEQ ID NO: 91 or 92 at the 3′ end of the reading frame of the DR5construct, wherein the tag is expressed at the C-terminal end of thepolypeptide. Clarified lysate is passed over 6 ml of Ni-NTA Agarose(Qiagen, Cat No. 30210) equilibrated with lysis buffer. The column iswashed with 20 ml of lysis buffer followed by 15 ml of PBS containing 50mM imidazole and 0.1% Triton X-100. DR5 ECD protein is then eluted with10 ml PBS containing 100 mM imidazole and 0.1% Triton X-100 followed bythe same buffer containing 250 mM imidazole. Fractions are checked on4-12% NuPAGE Bis-Tris gel (Invitrogen, Cat No. NP0329BOX). Positivefractions containing the DR5 ECD protein are pooled, concentrated andstored at −20° C.

After nickel chelate affinity chromatography, the protein is furtherpurified by size exclusion chromatography done on Amersham AKTA explorerwith software program Unicorn. The column is HiPrep 26/60 SephacrylS-200 HR with bed volume of 320 ml (Amersham, Cat No. 17-1195-01). Thestandard parameters are used according to manufacture's recommendations.Briefly, 1×PBS buffer (pH7.2) flew through the column at 0.5 ml/min, and0.5 ml/fraction is collected at 4° C.

The last step of purification is ion exchange chromatography. Theanion-exchanger Mono Q 5/50 GL column (Amersham, Cat No. 17-5166-01) isused with gradient elution at flow rate 1 ml/min, and 0.5 ml/fraction iscollected at 4° C.

1.1.4. Expression and Purification of DR5ECD-Fc Fusion Protein fromHEK293 Cells

Both human and cyno, long form and short form DR5 ECD are cloned intopRS5a-IgG expression vector containing a CMV promoter and a human IgG1Fc gene fragment. Positive clones are identified by PCR and confirmed byDNA sequencing. HEK293 cells are transient transfected withLipofectamine 2000 (Gibco, lot no. 11317078). The medium containingDR5ECD-Fc fusion protein is collected seven days post transfection. Theconcentrated supernatant is adjusted to pH 7.2, clarified by filtrationand loaded onto a 5 ml Protein A-Sepharose FF column at 0.5 ml/min.After baseline washing with PBS, pH 7.3, bound material is eluted with50 mM Citrate/140 mM NaCl, pH 2.7, neutralized and sterile filtered.

1.2. Immunizations

Three llamas are immunized with human DR5 antigen. One llama (106) isimmunized with the short (133 residues) recombinant human DR5(Peprotech, Rocky Hill N.J., catalog #310-19). Two other llamas areimmunized with full ectodomain human DR5-thioredoxin fusion. The llamasreceives seven weekly doses, injected intramuscularly of 50-100microgram of antigen (with Stimune (Cedi Diagnostics, Lelystad NL) as anadjuvant), followed by an additional 50 microgram dose two weeks later.Immune blood samples are taken at day 47 and 88 after the start of theimmunizations as well as lymph node tissues at day 88.

1.3. Library Constructions

cDNA samples are made from total RNA preparations of the immune bloodand lymph node samples. Nucleotide sequences encoding DR5 NB constructsare amplified from the cDNA samples of the three llamas immunized withhuman DR5 in a one-step RT-PCR reaction using primers ABL051 (SEQ ID NO:73), ABL052 (SEQ ID NO: 74) and ABL003 (SEQ ID NO: 75). Primer sequencesare shown in Table 5. The 700 by amplicons amplified from the IgG2 andIgG3 cDNA's in the sample are isolated from gel and subsequently used astemplate in a nested PCR reaction using the ABL050-MfeI primer (SEQ IDNO: 76) containing SfiI and MfeI restriction sites and the ABL003primer. The PCR products are subsequently digested with SfiI and BstEII(naturally occurring in FR4 of V_(HH) genes) and ligated into thecorresponding restriction sites of phagemid vector pAX50 to obtain alibrary after electroporation in Escherichia coli TG-1. pAX50 is anexpression vector derived from pUC119 which contained the LacZ promoter,a coliphage pill protein coding sequence, a resistance gene forampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the NB construct coding sequence, the vectorcoded for a C-terminal c-myc tag and a (His)6 tag. The phagemid vectorallows for production of phage particles, expressing the individual DR5NB constructs as a fusion protein with the gene3 product.

TABLE 5  Primer and linker sequences, with SEQ ID NO Name Sequences (5′to 3′) ID ABL051 GGCTGAGCTG GGTGGTCCTG G 73 ABL052GGCTGAGTTT GGTGGTCCTG G 74 ABL003 GGTACGTGCT GTTGAACTGT TCC 75 ABL050-CATTTGAGTT GGCCTAGCCG GCCATGGCAG 76 MfeI AGGTGCAATT GGTGGAGTCT GGGGGM13Fwd TGTAAAACGA CGGCCAGT 77 M13Rev CAGGAAACAG CTATGACC 78 Rev_TCAGTAACCT GGATCCCCCG CCACCGCTGC 79 30GlySerCTCCACCGCC GCTACCCCCG CCACCGCTGC CTCCACCGCC TGAGGAGACG GTGACCTG For_AGGTTACTGA GGATCCGGCG GTGGAGGCAG 80 GlySer35CGGAGGTGGG GGCTCTGGTG GCGGGGGTAG CGAGGTGCAG CTGGTGGAGT CTGG DR5 ECDCTGATCACCC AACAAGACCT AG 83 For DR5 ECD GCCTGAGAGA GAACAGGGAG A 84 RevDR5 cyno CACCATGGAA CAACGGGGAC AGAACGCC 85 For DR5 cynoTTAGGACATG GCAGAGTCTG CATTACCTTC 86 Rev

1.4. Selections

Different concentrations between 0 and 1 microgram/ml short (182 aminoacids) human DR5-Fc fusion protein (R&D Systems, Minneapolis Minn.,catalogue #631-T2/CF), full-ectodomain human DR5-Fc fusion protein((human DR5 amino acids 56 to 213 fused with human IgG1 Fc) andbiotinylated short (133 residues) recombinant human DR5 (Peprotech,Rocky Hill N.J., catalog #310-19) are immobilized on plates andstreptavidin-coated plates, respectively. Blocking is done using PBSsupplemented with 1% casein. Phages prepared from the three abovementioned pAX50 libraries are added and incubated for 30 minutes (in PBSsupplemented with 0.1% casein and 0.1% tween20). Unbound phages arewashed away (with PBS supplemented with 0.05% tween20); bound phages areeluted by addition of trypsin (1 mg/ml in PBS) and 30 min incubation at37° C. Eluted phages are allowed to infect exponentially growing TG-1cells that are then plated on ampicillin containing LB agar plates.Phages prepared from selected outputs are used as inputs in a secondselection round on full-length human DR5-Fc fusion protein (human DR5amino acids 56 to 213 fused with human IgG1 Fc) and biotinylated short(133 residues) recombinant human DR5 (Peprotech, Rocky Hill N.J.,catalog #310-19) as described above.

Plasmid DNA of the round 1 and 2 selection outputs is prepared, digestedwith SfiI and BstEII, and the DNA fragments encoding anti-DR5 NBconstructs are ligated into pAX51 vector and transformed into TG-1competent cells. pAX50 is an expression vector derived from pUC119 whichcontained the LacZ promoter, a resistance gene for ampicillin orcarbenicillin, a multicloning site and the gen3 leader sequence. Inframe with the NB construct coding sequence, the vector coded for aC-terminal c-myc tag and a (His)6 tag. Carbenicillin resistant clonesare analyzed for the presence of insert and sequences of positive clonesare verified. TG-1 cells containing the DR5 NB constructs of interestare grown in TB medium supplemented with carbenicillin and induced byaddition of IPTG for expression. The expression is allowed to continuefor 4 hours at 37° C. After centrifugation, cell pellets are frozenovernight, then resuspended for 1 hour at 4° C. in PBS ( 1/10^(th) ofculture volume), again followed by centrifugation. The resultingsupernatant is used as periplasmic extract.

1.5. Screening

Periplasmic extracts (as described above) are analyzed for DR5 bindingby ELISA. 10-fold dilutions of periplasmic extracts are added to platescoated with short (133 residues) recombinant human DR5 (Peprotech, RockyHill N.J., catalog #310-19) or cynomolgus DR5-Fc fusion protein (cynoDR5 amino acids 56 to 213 fused with human IgG1 Fc) amino acids 56 to213, IgG1 NVTS) and incubated for 2 hours (in PBS supplemented with 0.1%casein and 0.1% tween20). Unbound periplasmic extracts are washed away(PBS supplemented with 0.05% tween20) and bound NB constructs aredetected using mouse anti-myc (Roche, Basel CH, catalogue #11667149001)followed by rabbit anti-mouse-alkaline phosphatase (Sigma, St. LouisMo., catalogue # A-1902).

In another set of experiments, cell lines expressing human DR5 (Colo205)or cynomolgus DR5 (CHO-K1 cells transfected with an expression vectorcarrying the full length cynoDR5 gene downstream of a CMV promoter) areexposed to the periplasmic extracts (resuspended in PBS supplementedwith 10% fetal calf serum). Binding of NB constructs to cell-bound DR5is detected using mouse anti-myc (monoclonal antibody, clone 9E10 ATCC(Teddington, UK) number CRL-1729), is produced in mice as ascites andpurified in-house using standard affinity chromatography) followed byanti-mouse IgG-phytoerythrin (Jackson ImmunoResearch Laboratories, WestGrove, Pa., catalogue #115-115-164). Dead cells are counterstained withTO-PRO-3 (Molecular Probes, Carlsbad Calif., catalogue # T3605). Resultsare shown in Table 6.

TABLE 6 Binding of monomeric NB constructs to DR5 by ELISA(ABS_(405 nm)) NB Construct Human Cyno 10F1 0.754 0.124 11D1 0.540 0.21311H6 0.552 0.133 4E6 0.838 0.313 7A12 0.812 0.341

Binding of NB constructs to DR5 is evaluated by surface plasmonresonance on a Biacore 3000 instrument. Specificity of binding isanalyzed by allowing dilutions of the periplasmic extracts to pass overa CM5 sensor chip coated with 270 RU short (133 residues) recombinanthuman DR5 (Peprotech, Rocky Hill N.J., catalog #310-19). Thedissociation phases are analyzed and corresponding off-rates (k_(off))are given in Table 7 and Table 8. Table 7 shows results for the bindingof monomeric NB constructs to cell-bound DR5 by FACS (mean countfluorescence). Table 8 shows results for off-rates as determined bySurface Plasmon Resonance. Sequences are provided in Tables 1-4.

TABLE 7 Binding of monomeric NB constructs to cell-bound DR5 by FACS NBconstruct Human Cyno 10F1 125 1626 11D1 919 417 11H6 723 292 4E6 9722868 7A12 1097 24516

TABLE 8 Off-rates (1/s) as determined by Surface Plasmon Resonance NBconstruct k_(off) (1/s) 10F1 2.34 × 10⁻⁰³ 11D1 4.44 × 10⁻⁰⁴ 11H6 2.12 ×10⁻⁰³ 4E6 9.54 × 10⁻⁰⁵ 7A12 1.90 × 10⁻⁰⁴

2. Example II 2.1. Trimerization

DNA fragments encoding anti-DR5 NB constructs are digested with MfeI andBstEII and cloned into pAX73, pAX74 and pAX75 vectors in frame withlinker sequences. These are transformed into TG-1 competent cells andkanamycin resistant clones are analyzed for the presence of insert andsequence verified. The resulting constructs are digested with SfiI,BpuAI and NotI and the NB construct containing fragments are then clonedthrough a four-point ligation into SfiI-NotI digested pAX51 vector.Positive carbenicillin resistant clones, i.e., those encoding trivalentCMYC-HIS6-tagged NB construct constructs (each NB construct buildingblock fused to the next by a linker sequence), are again sequenceverified. pAX73, pAX74, pAX75 and pAX83 are pUC-derived cloning vectorsthat contain a resistance gene for kanamycin or neomycin, multicloningsites and in frame with the NB construct coding sequence, these vectorsencode GlySer linker sequences.

2.2. Tetramerization

DNA fragments encoding anti-DR5 NB constructs are amplified by means ofPCR using M13Rev (SEQ ID NO: 78) and Rev_(—)30 GlySer (SEQ ID NO: 79) orFor_GlySer35 (SEQ ID NO: 80) and M13Fwd (SEQ ID NO: 77) respectively.Both Rev_(—)30 GlySer and For_GlySer35 encode linker sequences (primersequences in Table 5). The PCR products are digested with MfeI, BamHIand BstEII and both fragments are jointly cloned through a three-pointligation in pAX75 and in pAX83, in frame with vector encoded linkersequences. These are transformed into TG-1 competent cells and kanamycinresistant clones are analyzed for the presence of bivalent NB constructinsert and sequence verified. Positive bivalent clones are then digestedwith SfiI, BpuAI and NotI and the NB construct containing fragments arethen cloned into SfiI-NotI digested pAX51 vector. Positive carbenicillinresistant clones, encoding tetravalent NB construct constructs, each NBconstruct building block fused to the next by a linker sequence, areagain sequence verified.

2.3. Pentamerization

Synthetic genes encoding the fourth GlySer linker sequence and the fifthNB building block are ordered from GeneArt AG (Regensburg, Germany).These fragments are then digested with HgaI and NotI and ligated intoBstEII-NotI digested tetravalent NB constructs.

2.4. Small Scale Expression

TG-1 cells containing the multivalent anti-DR5 NB constructs of interestare grown in baffled shaker flasks containing TB medium plus 100 μg/mlCarbenicillin and induced by addition of 1 mM IPTG for expression. Theexpression is allowed to continue for 4 hours at 37° C. After collectingthe cells, periplasmic extracts are prepared and the HIS6-tagged NBconstructs are purified by immobilized metal affinity chromatography(HisTrap FF Crude, GE Healthcare) followed by gel filtrationchromatography (Superdex 75 HR16/10, GE Healthcare) in PBS.

3. Example III 3.1. Binding on Biacore

Biacore CM5 sensor chips are coated with short (182 amino acids) humanDR5-Fc fusion protein (R&D Systems, Minneapolis Minn., catalogue#631-T2/CF) or cynomolgus DR5-Fc fusion protein (amino acids 56 to 213,IgG1 NVTS). Different concentrations (1 to 100 nM) of monovalent andmultivalent anti-DR5 NB constructs are then floated over the chips for akinetic analysis of the binding interaction. All multivalent anti-DR5 NBconstructs bind to human and cynomolgus DR5 (Table 9).

TABLE 9 Affinities (M) of anti-DR5 NB constructs as determined bysurface plasmon resonance Human DR5 Cynomolgus DR5 Clone Mono Tri TetraMono Tri Tetra 11D1 1.0 × 10⁻⁹ <3.0 × 10⁻¹¹ ND 2.2 × 10⁻⁷ <2.0 × 10⁻¹⁰ND 7A12 7.3 × 10⁻⁹ <3.0 × 10⁻¹² ND 7.1 × 10⁻⁹ <3.0 × 10⁻¹¹ ND 10F1 4.2 ×10⁻⁹ <5.0 × 10⁻¹¹ 3.9 × 10⁻¹² 1.4 × 10⁻⁷ <6.0 × 10⁻¹⁰ 2.2 × 10⁻¹¹ 11H66.7 × 10⁻⁹ <2.0 × 10⁻¹⁰ ND 9.2 × 10⁻⁶ <3.0 × 10⁻¹⁰ ND 4E6 1.7 × 10⁻¹⁰<5.0 × 10⁻¹¹ ND 1.6 × 10⁻⁸ <9.0 × 10⁻¹¹ ND ND: Not determined; BDL:below detection limit

3.2. Binding on FACS

In another set of experiments, cell lines expressing human (Colo205) orcynomolgus (CHO-K1 cells transfected with an expression vector carryingthe full length cynoDR5 gene downstream of a CMV promoter) DR5 areexposed to 1 nM of multivalent anti-DR5 NB constructs (resuspended inPBS supplemented with 10% fetal calf serum). Binding of NB constructs tocell-bound DR5 is detected using mouse anti-myc (monoclonal antibody,clone 9E10 ATCC (Teddington, UK) number CRL-1729, is produced in mice asascites and purified in-house using standard affinity chromatography)followed by anti-mouse IgG-phytoerythrin (Jackson ImmunoResearchLaboratories, West Grove, Pa., catalogue #115-115-164). Dead cells arecounterstained with TO-PRO-3 (Molecular Probes, Carlsbad Calif.,catalogue # T3605). All multivalent NB constructs bind specifically tocell bound human and cynomolgus DR5. Binding of the NB agents to each ofthe short form and the long form of DR5 is roughly equivalent for each,suggesting that all epitopes are held in common between the two forms

In another experiment, apparent binding constants are estimated fromsaturation binding experiments on FACS (as described above) of 10 to0.005 nM tetravalent anti-DR5 NB constructs to cell bound human andcynomolgus DR5. Apparent binding constants are calculated using GraphPadPrism 5 (GrafPad Software, San Diego, Calif.).

3.3. DR5-Specificity

A group of TNF receptor superfamily members are selected for testing theDR5 specificity (Table 10). The extracellular domain of members of theTNF receptor superfamily are cloned into pBadThioTopo and transformedinto E. coli strain TOP10 (Invitrogen #C4040-03). The protein isexpressed according to the protocol of manufacturer. The cell lysatesare collected for ELISA screening. 25 μL of 20% BSA is added per 10 mLof lysate. His capture plates (Sigma #S-5688) are coated with 100 μL oflysate and incubated 1 hour at room temperature. The remaining solutionis aspirated and wells are washed three times with PBS+0.05% Tween(PBST). The primary antibodies are diluted as follows in Blocking Buffer(PBS containing 10% FBS); LBY135 (positive control, anti-DR5 antibody,Novartis AG) is diluted to 0.24 mg/mL and rabbit anti-V5 (Abcam#Ab9116-100) is diluted 1:5000. 100 μL of diluted primary antibody isadded per well. Assay plates are incubated at room temperature for onehour and then washed three times with PBST. The secondary antibodies(goat anti-rabbit IgG-HRP) (Jackson Immunoresearch Laboratories#111-035-046) for wells incubated with V5, and goat anti-human IgG-HRP(Jackson Immunoresearch Laboratories #109-035-098) for wells incubatedwith LBY135) are diluted 1:5000 in PBS containing 5% FBS and 0.025%Tween. 100 μL of secondary antibody is added to appropriate wells. Afterincubation at room temperature for 1 hr, the plates are washed threetimes with PBST. 100 μL of Sure Blue (KPL #52-00-03) is added and oncethe color had developed, the plates are read at an absorbance of 650 nm.

TABLE 10 TNF receptor superfamily members TNFR TNFR Accession numbername Number ECD TNFRSF1A TNFR1 NM001065 Leu34-Thr211 TNFRSF1B TNFR2M32315 Thr27-Leu241 TNFRSF3 TNFR3 L04270 Pro30-Thr224 TNFRSF4 OX40X75962 Gly33-Gly212 TNFRSF5 CD40 X60592 Val18-Leu192 TNFRSF6 FAS M67454Ser20-Gly175 TNFRSF6B DcR3 AF104419 Val27-Ala176 TNFRSF7 CD27 M63928Thr21-Ser187 TNFRSF9 CD137 L12964 Glu19-Ile188 TNFRSF10C DcR1 AF012536Thr27-Leu241 TNFRSF10D DcR2 AF029761 Thr57-Thr198 TNFRSF11B OPG U94332Lys17-Ile197 TNFRSF13 BCMA NM001192 Met1-Thr56 TNFRSF13B TACI NM012452Met2-Val161 TNFRSF13C BAFFR NM052945 Met1-Gly64 TNFRSF16 NGFR M14764Lys29-Ile233

All NB constructs specifically bind to DR5, but not to other TRAIL andTNF receptor family members. See, e.g., FIG. 1.

3.4. Biacore Epitope Mapping

Biacore off-rate screening (Table 8) on short (133 residues) recombinanthuman DR5 (Peprotech, Rocky Hill N.J., catalog #310-19) revealed thatall anti-DR5 NB constructs bind to the extracellular domain of DR5 butnot to the 29 residue alternatively spliced region (amino acid residues185 to 231).

In another experiment, Biacore CM5 sensor chips are coated withmonovalent anti-DR5 NB constructs (230 to 520 RU). Then, 500 nM of short(133 residues) recombinant human DR5 (Peprotech, Rocky Hill N.J.,catalog #310-19) is floated over the chips until saturation is reached.At this point, 50 nM human TRAIL (R&D Systems, Minneapolis Minn.,catalogue #375-TEC/CF) is floated over the chips and the increase inbinding signal is used to categorize the NB constructs in differentepitope classes compared to the TRAIL epitope (Table 11).

TABLE 11 Epitope mapping of anti-DR5 NB constructs compared to TRAIL NBconstruct Result 11D1 No TRAIL blocking 4E6 Partial TRAIL blocking 10F1TRAIL blocking 11H6 No TRAIL blocking 7A12 TRAIL blocking

3.5. In Vitro Cell Survival

Cells are maintained in logarithmic growth phase prior to theexperiments. On the day of the assay, cells are transferred to 96-wellplates (Corning Inc., Lowell, Mass., Cat#3917) at 5,000-20,000 cells perwell, then series diluted anti-DR5 NB constructs are added at 50μl/well. After incubation for 24 to 72 hours, the relative number ofsurviving cells are quantified using a luciferase-based ATPquantification Kit (Cell Titer Glo, Promega, Madison, Wis. Cat#G7571)and read on a luminescence plate reader (Fluoroskan Ascent Fla., ThermoElectron, Waltham, Mass.).

A panel of trivalent anti-DR5 NB constructs is screened in a cellsurvival assay with Colo205 cells. All trivalent constructs inducedapoptosis on Colo205 (see Table 12) in contrast to their monovalentcounterparts (data not shown) that are not biologically active in thisassay. A tetravalent construct of an irrelevant (non-DR5 binding) NBconstruct did not induce apoptosis.

TABLE 12 In vitro potency (IC₅₀ (M)) of trivalent anti-DR5 NB constructson Colo205 NB construct IC₅₀ (M) 10F1 tri 4.07 × 10⁻¹¹ 11D1 tri 1.11 ×10⁻¹⁰ 11H6 tri 4.58 × 10⁻¹¹ 4E6 tri 3.79 × 10⁻¹¹ 7A12 tri 1.20 × 10⁻¹⁰

In another experiment (Table 13), selected trivalent anti-DR5 NBconstructs are screened in cell survival assays against a panel of tumorand normal cell lines.

TABLE 13 In vitro potency (IC₅₀ (M)) of trivalent anti-DR5 NB constructs4E6 tri 11D1 tri 10F1 tri 7A12 tri 11H6 tri Jurkat 20 20 20 20 20 Molt420 20 20 20 20 A549 20 20 20 20 20 H226 2.0 × 10⁻¹¹ 3.0 × 10⁻¹¹ 2.0 ×10⁻¹¹ 3.0 × 10⁻¹¹ 20 H2052 6.0 × 10⁻¹² 1.5 × 10⁻¹¹ 1.3 × 10⁻¹¹ 6.4 ×10⁻¹¹ 3.6 × 10⁻¹⁰ H2122 2.0 × 10⁻¹² 4.0 × 10⁻¹² 3.0 × 10⁻¹² 8.0 × 10⁻¹²1.2 × 10⁻¹¹ M30 20 20 20 20 20 Panc-1 20 20 20 20 20 MiaPaCa-2 2.3 ×10⁻¹¹ 20 20 20 20 ARPE-19 20 20 20 20 20 IMR-90 20 20 20 20 20 Huvec 2020 20 20 20 20: IC₅₀ 20 nM

In another experiment (Table 14), selected tetravalent anti-DR5 NBconstructs are screened in cell survival assays against a panel of tumorand normal cell lines.

TABLE 14 In vitro potency (IC₅₀ (M)) of tetravalent anti-DR5 NBconstructs 4E6 tetra 11D1 tetra 10F1 tetra 7A12 tetra 11H6 tetra Colo2058.0 × 10⁻¹³ 8.1 × 10⁻¹² 2.0 × 10⁻¹² 6.6 × 10⁻¹² 3.7 × 10⁻¹² Jurkat 6.0 ×10⁻¹³ 3.5 × 10⁻¹¹ 3.0 × 10⁻¹² 3.7 × 10⁻¹¹ 9.1 × 10⁻¹² Molt4 <1.0 ×10⁻¹³  1.5 × 10⁻¹¹ 2.0 × 10⁻¹² 20 8.0 × 10⁻¹³ A549 3.3 × 10⁻¹⁰ 20 20 2020 H2122 8.0 × 10⁻¹³ 8.0 × 10⁻¹² 1.0 × 10⁻¹² 3.6 × 10⁻¹² 2.8 × 10⁻¹²H226 1.0 × 10⁻¹¹ 8.9 × 10⁻¹¹ 2.4 × 10⁻¹¹ 20 1.1 × 10⁻¹⁰ H2052 8.0 ×10⁻¹³ 2.5 × 10⁻¹¹ 4.0 × 10⁻¹² 3.3 × 10⁻¹¹ 1.5 × 10⁻¹¹ M30 20 20 20 20 20Panc-1 3.0 × 10⁻¹² 20 9.0 × 10⁻¹² 20 2.2 × 10⁻¹⁰ MiaPaCa-2 1.8 × 10⁻¹²1.9 × 10⁻¹¹ 2.8 × 10⁻¹¹ 4.1 × 10⁻¹¹ 1.1 × 10⁻¹¹ BxPC-3 1.9 × 10⁻¹² 4.1 ×10⁻¹¹ 3.0 × 10⁻¹² 2.3 × 10⁻¹¹ 2.7 × 10⁻¹¹ Malme-3 20 20 20 20 20 WI-3820 20 20 20 20 ARPE-19 20 20 20 20 20 184A1 20 20 20 20 20 Huvec 20 2020 20 20 HAAE-1 20 20 20 20 20 20: IC₅₀ 20 nM

Graphic representations showing that increasing the hierarchy ofmultimers results in increased efficacy and potency is shown in FIGS.6A, 6B and 6C, wherein trimeric, tetrameric and pentameric 11H6 and 4E6NB constructs have increasing potency, respectively, against treatedColo205 and H226 tumor cells in vitro.

The multivalent anti-DR5 NB constructs display apoptosis-inducingactivity on tumor cell lines but not on normal (healthy) cell lines. Thegeneral trend is that increased apoptosis-inducing activity is seen withan increased number of subunits in a multimeric NB composition.Increasing from tetrameric to pentameric forms of the NB providesevidence of further increase in potency in cell death assays, asexemplified by lowering the IC50 or increasing the maximum cell deathachieved depending on the cell line.

3.6. Cross-Linking

In another experiment, multivalent NB constructs (10 micrograms) thatcontain a cMYC tag are cross-linked through their CMYC-tag with 1.5 μgup to 150 micrograms of mouse anti-myc (monoclonal antibody, clone 9E10ATCC (Teddington, UK) number CRL-1729) is produced in mice as ascitesand purified in-house using standard affinity chromatography) during a30 minute incubation at room temperature. The cross-linked NB constructsare then evaluated in in vitro cell survival assays. Increasedcross-linking correlated with improved potency and efficacy (see Tables15-16). Table 15 provides in vitro potency (IC50 (M)) of trivalentanti-DR5 NB constructs cross-linked with different amounts of antibodyon Jurkat cells. Table 16 provides in vitro efficacy (% dead cells) oftetravalent anti-DR5 NB constructs cross-linked with different amountsof antibody on Jurkat cells. The general trend is that more cells arekilled with increased amounts of cross-linking of the tagged NBmultimers.

TABLE 15 In vitro potency (IC₅₀ (M)) of cross-linked trivalent anti-DR5NB constructs Jurkat cells Weight ratio (μg/μg) IC₅₀ (M) 11D1 tri 0 Noapoptosis observed 11D1 tri 0.15 1.1 × 10⁻⁸  11D1 tri 15.0 7.3 × 10⁻¹⁰Ratio: Weight ratio of cross-linking antibody/tetravalent anti-DR5 NBconstructs

TABLE 16 In vitro efficacy (% dead cells) of cross- linked tetravalentanti-DR5 NB constructs Jurkat cells Weight ratio (μg/μg) Efficacy (%dead cells) 11D1 tetra 0 48 11D1 tetra 0.15 48 11D1 tetra 0.75 60 11D1tetra 1.5 63 11D1 tetra 7.5 88 11D1 tetra 15.0 93 Ratio: Weight ratio ofcross-linking antibody/tetravalent anti-DR5 NB constructs

3.7. Serum Stability

3.7.1. NB Constructs and DR5 Binding in ELISA

NB constructs are pre-incubated with 100% fresh human serum at 37° C.for 24 hours. Flat bottom 96-well ELISA plates (Costar, Cambridge,Mass., Catalog No. 3590) are coated overnight at 4° C. with 50 μl DR5-Fcfusion protein (DR5 amino acids 56 to 213) at 1 μg/ml in PBS. The platesare then blocked with 300 μl 2% BSA (Gibco, Grand Island, N.Y.,Cat#11018-025) in PBS for one hour and washed three times. NB constructsamples are serially diluted in the PBS buffer containing 10% humanserum, then added in ELISA plates and incubated for one hour at roomtemperature. After wash, 1:5000 HRP conjugated anti-myc polyclonalantibody (Invitrogen: 460709) or 1:7000 rabbit anti-V_(HH) antibodyfollowed by 1:15000 HRP conjugated anti-rabbit IgG (JacksonImmunoResearch Laboratories, Inc. West Grove, Pa., Cat#115-035-072) isadded in plates. After a final set of washes, 100 μl/well of TMBMicrowell peroxidase substrate (BioFX, Owings Mills, Md., Catalog No.TBNP-1000-01) is added and incubated at room temperature for 10 minutes.The reaction is stopped with 100 μl Stop Reagent (BioFX, Owings Mills,Md., Cat#LSTP-0100-01). Colorimetric detection of the product is done at450 nm in SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale,Calif.). Data is analyzed with SoftMax Pro software (Molecular Devices,Sunnyvale, Calif.) according to its user's manual.

Human serum shows little interference with NB construct trimers ortetramers binding to DR5, detected by either anti-Myc or anti-NBconstruct-antibody.

3.7.2. NB Construct Function in Cell Survival Assay

Cell survival assays are also performed in presence and absence of about10-15% human serum albumin. Tetravalent anti-DR5 NB constructs maintaintheir in vitro potency compared to control assays in the presence of 10%fetal calf serum.

4. Example IV 4.1. Single Dose In Vivo Trivalent Anti-DR5 NB Constructs

Outbred athymic (nu/nu) female mice (Harlan Sprague Dawley,Indianapolis, Ind.) are anesthetized and implanted s.c. into the rightaxillary (lateral) region of each animal with 1×10⁶ Colo205 cells.Tumors are allowed to grow until the size of 200 mm³ before the dosingis initiated. NB construct is formulated as a solution in PBS andadministered single dose at 200 ug/mouse by a bolus i.v. injection.Tumor samples are collected from groups of animals (one group per timepoint, 3 animals per group) at 1 h, 2 h, 4 h and 8 h.

Part of the tissue samples are used for immunohistochemical (IHC)staining of Caspase3. The tissues are immediately put into 10% neutralbuffered formalin (NBF, Fisher Scientific, Pittsburgh, Pa., Catalog No.SF100-20), processed and embedded into paraffin blocks. Sections of 4.0μm are cut on to SuperFrost Plus charged slides (Fisher Scientific,Catalog No. 12-550-15). The sections are deparaffinized in xylene (5minutes×2) and hydrated in a graded series of alcohols (100% alcohol for5 minutes, 95% alcohol for 2 minutes, 70% alcohol for 2 minutes, andde-ionized water for 5 minutes). The hydrated sections underwent antigenretrieval which is conducted by microwaving the slides in citric acidbuffer (0.01M, pH 6.0, BioGenex, San Ramon, Calif., Catalog No.HK080-9K) for 10 minutes. The slides are placed onto the staining racksof the autostainer (Dako Cytomation) after they are washed withde-ionized water for 5 minutes. The following steps are done with theDako automated system at room temperature. Between each steps, theslides are rinsed with the rinse buffer (OptiMax Wash Buffer, Biogenex,Catalog No. HK583-5K) three times. Endogenous peroxidase is blocked byincubation of the slides with hydrogen peroxide (Dako, Catalog No.S2001) for 10 minutes. The nonspecific antigen reaction is blocked bythe Block Serum (Dako, Catalog No. X0909) for 10 minutes. The slides areincubated with the cleaved Caspase3 rabbit polyclonal antibody (CellSignaling Technology, Beverly, Mass., Catalog No. 9661) for 60 minutes,then rinsed and exposed to the peroxidase conjugated antibody (DAKOEnvision system, Catalog No. K4003) for 30 minutes. For detection, DAB(3,3′-diaminobenzidine) solution (Dako Cytomation, Catalog No. K3468) isapplied for 2 minutes. Immunostained slides are lightly counterstainedwith Harris Hematoxylin (SurgiPath, Catalog No. 01560), dehydratedthrough a series of increasing alcohol concentrations (70%, 95% and 100%alcohol, each for 1 minute) and xylene (5 minutes X2), and mounted witha coversliper (Leica).

The slides are scanned into the digitized slide database using theAperio system (Aperio Technologies, Inc. Vista, Calif.) and imageanalysis is done by using the optimized Positive Pixel CountingAlgorithm from Aperio. The whole sections excluding necrotic areas andhost tissue portion “marked out” by using the tools provided by theimaging system are used for image analysis. All multivalent anti-DR5 NBconstructs induce Caspase3 activation.

Part of the tissue samples are mixed with T-per buffer (Pierce,Rockford, Ill., Catalog No. 78510) then immediately homogenized in thetissue lyser (Qiagen, Catalog No. 85210). The supernatant of tissuelysate is collected and its protein concentration is determined using astandard BCA assay (Pierce, Catalog No. 1856210). The tissue lysates areused in the following assays to check NB construct and antibodyconcentration plus Caspase3 activity in tumor.

NB construct concentration is determined in ELISA. The ELISA plates(Nunc, Rochester N.Y., Catalog No. 439454) are coated overnight at 4° C.with 100 μl human DR5-Fc fusion protein (DR5 amino acids 56 to 213) at 1μg/ml in PBS, then blocked with 1% BSA (Gibco, Catalog No. 11018-025) inPBS for 1 hour at room temperature and then washed three times. Theserially diluted serum or tissue lysate samples are transferred intoELISA plates along and incubated 1.5 hours at room temperature.Following three washes, 1/22500 diluted anti-V_(HH) is added to theELISA plates, and incubated for one hour at room temperature. The platesare washed three more times and then incubated for one hour with 100μl/well of 1/20000 diluted HRP-goat-anti-rabbit (Pierce, Catalog No.31462). After a final set of washes, 100 μl/well of TMB Microwellperoxidase substrate (BioFX, Owings Mills, Md., Catalog No.TMBW-0100-01) is added for 10 minutes and the reaction is then stoppedwith 100 μl stop solution (BioFX, Catalog No. LSTP-1000-01).Colorimetric detection of the product is done at 450 nm in SpectraMax M5microplate reader (Molecular Devices, Sunnyvale, Calif.). Data isanalyzed in 4-parameter model with SoftMax Pro software (MolecularDevices, Sunnyvale, Calif.) according to its user's manual. All NBconstructs show quick clearance in serum and in tumor tissue, and fasttissue penetration.

Caspase 3/7 activity in tumor tissue lysate is evaluated by Caspase gloassay (Promega Catalog no. G8092) according to its user's manual.Luminescence is read using the SpectraMax M5 microplate reader(Molecular Devices, Sunnyvale, Calif.). Consistent with IHC results, allNB constructs activate Caspase 3 within 8 hours.

4.2. Single Dose In Vivo Tetravalent Anti-DR5 NB Constructs

Outbred athymic (nu/nu) female mice (Harlan Sprague Dawley,Indianapolis, Ind.) are anesthetized and implanted s.c. into the rightaxillary (lateral) region of each animal with 1×10⁶ Colo205 cells.Tumors are allowed to grow until the size of 200 mm³ before the dosingis initiated. NB construct is formulated as a solution in PBS andadministered single dose at 200 ug/mouse by a bolus i.v. injection.Serum and tumor tissue are collected from groups of animals (one groupper time point, 3 animals per group) at the following time points: 0.25h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h. The tumor tissue sample ismixed with T-per buffer (Pierce, Rockford, Ill., Catalog No. 78510) thenimmediately homogenized in the tissue lyser (Qiagen, Catalog No. 85210).The supernatant of tissue lysate is collected and its proteinconcentration is determined using a standard BCA assay (Pierce, CatalogNo. 1856210).

NB construct concentration in serum and tissue lysate is determined inELISA as described above. Mean serum concentration-time profiles aresubjected to a two-compartmental pharmacokinetic analysis using model 7within WinNonlin Professional Software Version 5.1 (PharsightCorporation, Mountain View Calif., USA). For each NB construct theconcentration-time profile is fit using iterative reweighting 1/y.y,where y is the predicted concentration. The mean residence time (MRT,Table 17) off the different NB constructs ranged from 2.6 to 13.5 hr.The Volume of distribution (Vss) of the different NB constructs is 2-35times larger than the plasma volume of a 20 g mouse (≈1.5 ml),indicating extensive distribution outside the vascular space. Theoverall clearance rate of the different NB constructs is at least 5times less than the murine glomerular filtration rate (≈15 ml/hr),hence, all tetravalent constructs are highly effective in reducingsignificantly the urinary filtration and subsequent excretion at thelevel of the kidney.

TABLE 17 PK-parameters of tetravalent anti-DR5 NB constructs Parameter10F1 tetra 11D1 tetra 11H6 tetra 4E6 tetra 7A12 tetra C₍₀₎ (μg/ml) 114336 163 25.2 71.7 V_(ss) (mL) 12.5 5.63 2.83 56.8 12.2 V₁ (mL) 1.760.595 1.22 7.95 2.79 V₂ (mL) 10.7 5.04 1.61 48.8 9.43 CL (mL/hr) 2.852.11 0.211 4.2 1.42 CL_(d) (mL/hr) 1.29 0.453 0.387 2.89 1.59 t_(1/2α)(hr) 0.3 0.161 1.04 0.756 0.589 t_(1/2β) (hr) 8.49 9.41 11.1 20.3 9.46MRT (hr) 4.38 2.68 13.4 13.5 8.57 AUC_(inf) 70.2 95 947 47.6 140 (μg*hr/ml)

Caspase 3/7 activity in tumor tissue lysate is evaluated by Caspase gloassay (Promega Catalog no. G8092) according to its user's manual anddescribed as above. All tetramers have similar PD profile (FIG. 2).

An indirect response model is developed to describe the potency (EC₅₀),efficacy (E_(max)) and time-course of the pharmacological effect of thedifferent NB constructs. The model describes a change in cellularcaspase level that results from an enhanced production rate followingDR5 receptor binding (i.e., stimulation of the build-up of cellularcaspase). The rate of cellular caspase (Response, R) is described by thefollowing equation.

$\begin{matrix}{\frac{R}{t} = {{k_{i\; n}{\bullet \left\lbrack {1 + \frac{E_{\max}\bullet \; C^{n}}{{EC}_{50}^{n} + C^{n}}} \right\rbrack}} - {k_{out}\bullet \; R}}} & \left( {{equation}\mspace{14mu} 1} \right)\end{matrix}$

where k_(in) is the zero-order synthesis rate, R is the caspase level,E_(max) is the maximum stimulation, C is the NB construct concentrationat the tumor, n is the response shape factor and k_(out) is the caspasefirst order elimination rate constant.

The tumor concentration-time profiles are first fitted to thepharmacokinetic function that is minimally necessary to provide areasonable characterization of the concentration-time data(two-compartmental model 3 or model 11 within WinNonlin Software Version5.1). The pharmacokinetic function obtained for each NB construct isthen used as input function for equation 1. The obtained pharmacodynamicparameters are listed in Table 18.

TABLE 18 PK/PD-parameters single dose tetravalent anti-DR5 NB constructsParameter¹ 10F1 tetra 7A12 tetra 4E6 tetra 11D1 tetra 11H6 tetra k_(out)(1/hr) 0.319 0.301 0.412 0.416 0.356 EC₅₀ (ng/ 0.365 1.41 1.36 12.2 8.37mg tissue) E_(max) 15.8 15.3 4.74 12.2 17.5 N 2.14 3.85 10.1 0.18 4.62

4.3. In Vivo Test Using Xenograft Model

Outbred athymic (nu/nu) female mice (Harlan Sprague Dawley,Indianapolis,

IN) are implanted s.c. into the right axillary (lateral) region withapproximately 1-2×10⁶ COLO205 tumor cells suspended in 100% Hanks'balanced salt solution (HBSS) in a total volume of 100 ml. Tumors areallowed to grow to 200 mm³ before the dosing is initiated. NB constructsare formulated as a solution in PBS, and dosed either once per week orthree times per week on Monday, Wednesday and Friday. Both treatmentsare administered i.v. for four weeks. Tumors are measured and individualanimal body weights are recorded once or twice per week. Experiments areconcluded after seven weeks from the initial dosing. Anti-tumor activityis expressed as % T/C (comparing change in tumor volume for treatmentgroup versus vehicle control group). Regression is calculated using theformula: (1−T/T0)×100%, where T is the mean tumor volume for thetreatment group at the end of the experiment, and T0 is the mean tumorvolume at the beginning of the experiment. Statistical significance ofthe results is uniformly evaluated using one-way ANOVA test post-hocTukey analysis. As shown in FIG. 3, the NB constructs induce tumorstasis or regression in the Colo205 tumor model. Pentameric forms of theNB constructs trend toward more potent efficacy than tetrameric forms.

5. Example V 5.1. Humanization of 4E6

5.1.1. Characterization of 4E6 Humanization Variant

The protein sequence of parent 4E6 is aligned to the human VH3-23(DP-47) and JH5 germlines (Table 19). Amino acid differences relative tothe human germline sequence are represented by letters, identical aminoacids by dots. Amino acid differences in framework regions that areunderlined are selected for conversion into the human counterpartwhereas the others are left untouched.

TABLE 19 Alignment of 4E6 parent and humanized N-glycosylation knock out variantsKabat #: 1        10        20        30        40        50         60|--------|---------|---------|---------|---------|--a-------|VH3-23/JH5:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYA 4E6:....v......s..a.d.........r..g.irvg.f..t...er.f.a..nrnd.t.... 4E6-hu:..........................r..g.irvg.f ......r.f....nrnd.t.... Kabat #:60        70        80          90         100                110 |---------|--------|--abc-------|----------|abcdefghi---------|---VH3-23/JH5: DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK------------------WGQGILVIVSS 4E6: .............a...v.m..a..kp..........aglqy

adrvpvgavy.....q..... 4E6-Hu: .................v........p..........aglgynrAadrvpvgavy...........VH3-23/JH5 = SEQ ID NO: 90 4E6 = SEQ ID NO: 1 4E6-Hu = SEQ ID NO: 26

Purified, monovalent material is produced from 4E6, 4E6-Hu and ofvariant 4E6hx (wherein 4E6hx has the humanized FR regions identical to4E6-Hu but has the CDR3 of 4E6). These constructs are then characterizedin a number of assays for binding on human and cynomolgus DR5, namelyFACS saturation binding (apparent Kd) and SPR kinetics (see Table 20).In addition, the variants are analyzed in the thermal shift assay aswell (Table 20). 5 μl of each purified monovalent anti-DR5 NB construct(80 μl/ml) is incubated with 5 μl of the fluorescent probe Sypro Orange(Invitrogen, Carlsbad, Calif., catalogue # S6551) (final concentration10×) in 10 μl of buffer (100 mM phosphate, 100 mM borate, 100 mMcitrate, 115 mM NaCl, buffered at different pH's ranging from 3.5 to 9).The samples are then heated in a LightCycler 48011 machine (Roche,Basel, Switzerland), from 37° C. to 90° C. at 4.4° C./s, after whichthey are cooled down to 37° C. at 2.2° C./s. Upon heat-inducedunfolding, hydrophobic patches of the proteins are exposed, to which theSypro Orange binds resulting in an increase in fluorescence intensity.The inflection point of the first derivative of the fluorescenceintensity curve serves as a measure of the melting temperature (Tm). Formore details, please see Ericsson et al. 2006 (Annals of Biochemistry,357: 289-298). Overall, the 4E6-hx variant displays affinities similarto the 4E6 parent. In addition, this variant has a 7% increase in Tmcompared to the 4E6 parent.

TABLE 20A Monovalent 4E6 humanization and N- glycosylation knock-outvariants Human DR5 FACS Tm @ Biacore Apparent pH k_(a) (1/Ms) k_(d)(1/s) KD (M) KD (M) 7(° C.) 4E6 4.4 × 10⁺⁰⁶ 1.0 × 10⁻⁰⁴ 2.3 × 10⁻¹¹ 4.2× 10⁻¹⁰ 73.4 4E6- 6.0 × 10⁺⁰⁶ 1.4 × 10⁻⁰⁴ 2.3 × 10⁻¹¹ 3.4 × 10⁻¹⁰ 79.1hx 4E6- 1.1 × 10⁺⁰⁷ 1.1 × 10⁻⁰⁴ 9.5 × 10⁻¹² 4.4 × 10⁻¹⁰ ND Hu

TABLE 20B Monovalent 4E6 humanization and N- glycosylation knock-outvariants Cynomolgus DR5 FACS Tm @ Biacore Apparent pH k_(a) (1/Ms) k_(d)(1/s) KD (M) KD (M) 7(° C.) 4E6 2.1 × 10⁺⁰⁶ 1.1 × 10⁻⁰² 5.5 × 10⁻⁰⁹ 7.3× 10⁻⁰⁹ 73.4 4E6- 6.1 × 10⁺⁰⁶ 2.1 × 10⁻⁰² 3.4 × 10⁻⁰⁹ 7.1 × 10⁻⁰⁹ 79.1hx 4E6- 7.0 × 10⁺⁰⁶ 1.1 × 10⁻⁰² 1.6 × 10⁻⁰⁹ 8.8 × 10⁻⁰⁹ ND Hu

5.1.2. N-Glycosylation Knock-Out Variant of Humanized 4E6

Expression of 4E6-hx in P. pastoris followed by SDS PAGE and anti-V_(HH)(polyclonal anti-Nanobody®) western reveals the presence of two products(16 kDa and 21 kDa) compared to only one 16 kDa product when E. coli isthe expression host. Treatment with PNGaseF and staining withconcanavalin reveals that the 21 kDa product is the N-glycosylated formof the 16 kDa product. Cleavage of N-glycosylation moieties fromproteins using PNGaseF is done according to the manufacturer'srecommendations (New England Biolabs, Ipswich, Mass., catalogue #P0704S). PNGaseF is an amidase that cleaves between the innermost GlcNAcand asparagine residues of high mannose, hybrid, and complexoligosaccharides from N-linked glycoproteins. Detection of proteinN-glycosylation is done in a Western assay with Concanavalin A, a lectinthat recognizes glycoproteins containing α-D-mannose, α-D-glucose.Blotted proteins are blocked for 2 hours with blocking buffer (PBS,0.05% Tween-20, 1 mM CaCl₂, 1 mM MnCl₂), followed by ON incubation with5 μg/ml of Concanavalin A biotin conjugate (Sigma, St. Louis Mo.,catalogue # C2272) at 4° C. in blocking buffer. Detection is done usinga 1/2000 dilution of extravidin-HRP conjugate in blocking buffer and DABas substrate (Sigma, St. Louis Mo., catalogue # E2886 and D6815). The 36kDa band visible in the coomassie stained gel is the PNGaseF enzyme.Off-rate analysis on human DR5 revealed an >8-fold increase for the P.pastoris produced material versus the E. coli produced material,suggesting that the N-glycosylation interferes with the binding of4E6-hu to DR5.

A potential N-glycosylation motif is present in the CDR3 of 4E6 and4E6-hx (amino acids “nrs”—shown in bold and italics in Table 19). The4E6-Hu variant (CDR3 with a replaced “A” residue shown in bold capitalletter—Table 19) is generated and produced in E. coli, purified andassayed for binding to human and cynomolgus DR5 using the FACSsaturation binding assay and SPR kinetics (Table 20). The 4E6-Hu hasequal binding characteristics to both human and cynomolgus DR5, comparedto 4E6-hx. Upon expression in P. pastoris, the 4E6-Hu variant yieldsonly the 16 kDa product. This confirms the functional knock-out of theglycosylation motif.

5.1.3. In Vitro Characterization of 4E6-Hu

Purified, tetravalent material is produced from wild type 4E6 and itshumanized variant 4E6-Hu. These are then assayed in cell survival assaysin Colo205 and Jurkat (Table 21). The potency and efficacy of thehumanized variant is relatively equal to the parent 4E6 construct.

TABLE 21 In vitro potency and efficacy of tetravalent wild type 4E6 and4E6-Hu Colo205 Jurkat IC50 Efficacy IC50 Efficacy (M) (% dead cells) (M)(% dead cells) 4E6 tetra 1.9 × 10⁻¹¹ 94 8.7 × 10⁻¹² 79 4E6-Hu 1.2 ×10⁻¹¹ 94 2.6 × 10⁻¹² 95 tetra

5.2. 11H6 Humanization

5.2.1. Characterization of 11H6 Humanization Variant

The protein sequence of wild type 11H6 is aligned to the human VH3-23(DP-47) and JH5 germlines (Table 22). Amino acid differences relative tothe human germline sequence are represented by letters, identical aminoacids by dots. Amino acid differences in framework regions that areunderlined are selected for conversion into the human counterpartwhereas the others are left untouched.

TABLE 22  Alignment 11H6 parent and humanization variantKabat #:    1        10        20        30        40        50         60            |--------|---------|---------|---------|---------|--a-------|VH3-23/JH5: EVQLLESGGGLVQPGGSLRLSCAASGFTESSYAMSWVRQAPGKGLEWVSAISGSGGSTYYA11H6:       ....v.....................tfdkinn.g.y......qrdl.aq.t-p..d.d..11H6-hu:    ..........................tfdkinn.g.y......qrdl.aq.t-p..d.d..Kabat #:    60        70      80             90        100             110             |---------|-------|--abc---------|---------|abcdef---------|---VH3-23/JH5:   DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK--------------WGQGTLVTVSS11H6:         .............a.d.m.......kp......f.naeilkrayidvyvny.....q.....11H6-hu:      ..........................p....... naeilkrayidvyvny...........VH3-23/JH5 = SEQ ID NO: 90 11H6 = SEQ ID NO: 5 11H6-hu = SEQ ID NO: 30

Purified, monovalent material is produced for 11H6 and 11H6-Hu, which isthen characterized in a number of assays for binding on human andcynomolgus DR5, namely FACS saturation binding (apparent Kd) and SPRkinetics (Table 23). In addition, the variants are analyzed in thethermal shift assay (Table 23). The 11H6-hu variant retains the parental11H6 binding characteristics. It also has an 8% increase in meltingtemperature compared to parent 11H6. Tm is determined at pH 7.

TABLE 23 Characterization of monovalent 11H6 parent and humanizationvariants Apparent Kd (M) Kd (M) Cynomolgus Cynomolgus Tm Human DR5 DR5Human DR5 DR5 (° C.) 11H6 4.6 × 10⁻⁰⁹ ND 4.9 × 10⁻⁰⁹ 3.2 × 10⁻⁰⁷ 68.511H6- 4.4 × 10⁻⁰⁹ ND 2.3 × 10⁻⁰⁹ 1.7 × 10⁻⁰⁷ 74.0 hu

5.2.2. In Vitro Characterization of 11H6-Hu

Purified, tetravalent material is produced from parental clone 11H6 andits humanized variant 11H6-Hu. These are then assayed in cell survivalassays against Colo205 and Jurkat (Table 24). The potency and efficacyof the humanized variant is equal to the parent. Efficacy represents thepercentage of dead cells seen.

TABLE 24 In vitro potency and efficacy of tetravalent wild type 11H6 and11H6-hum Colo205 Jurkat IC50 (M) Efficacy IC50 (M) Efficacy 11H6 tetra1.4 × 10⁻¹¹ 92 1.5 × 10⁻¹¹ 34 11H6-hu tetra 2.0 × 10⁻¹¹ 94 1.6 × 10⁻¹¹54

5.3. In Vitro Characterization of 11H6-Hu and 4E6-Hu Tetramers andPentamers

Cells are plated in a 96 well plate (Costar #3903 white clear bottom)with the intention of achieving about 75% confluence. Cells are platedin a range from 7500-15000 cells/well (depending on cell line) in 100 μlmedia the day prior to the assay. The day of the assay, the media isremoved and fresh media is added. Serially diluted NB agent is thenadded (starting concentrations may vary depending on the cell line),usually between 1-20 nM starting concentration, which is then diluted3-4 fold over 10 dilutions in general for a final volume of 150 μl. Thecells are then incubated for 24-72 hrs at 37° C. in the CO₂ suppliedincubator. After incubation, the plated cells and Cell Titer Glo(Promega #G7571) reagents are brought to room temperature (about 30min). 50 μL of the reconstituted Cell Titer Glo reagent is then added toeach well. The plate is shaken for 2-3 minutes and then left toequilibrate in the dark for 10 minutes before reading luminescence onthe Spectromax.

Specific examples are provided in Table 25.

TABLE 25 In vitro characterization of 11H6-hu and 4E6-hu tetramers andpentamers 11H6-Hu 4E6-Hu fold fold improve- improve- IC50 (nM) tetrapenta ment tetra penta ment Jurkat (72) 0.0007 0.0017 4 0.0172 0.0036 5A549 >10 >10 1 >10 >10 1 Colo205 0.0036 0.0012 3 0.0034 0.0007 5 BxPC-30.0154 0.0046 3 0.0292 0.005 6 T24 0.0222 0.0031 7 0.0447 0.0043 10Panc-1 0.2317 0.0067 35 0.2291 0.0105 22 M30 >10 >10 1 >10 >10 1 H2260.1718 0.0124 14 0.1293 0.0272 5 H2122 0.0041 0.0017 2 0.0051 0.0008 7MiaPaCa- 0.0117 0.0031 4 0.0185 0.0038 5 2

6. Example VI 6.1. Ex-Vivo Assay for Leukemia Patients

An ex-vivo assay for leukemia patients is provided as a method topredict which patients will respond to treatment with an NB construct. Ablood sample is obtained from a patient prior to treatment and used toassay potential treatment response. The degree and direction of responseis used as selection criteria for treatment.

T-cell leukemia (T-ALL) is believed to be generally sensitive to DR5stimulation. T-ALL samples are treated with NB constructs of theinvention and assessed to determine if the number of leukemia cells isdecreased (or undergoing apoptosis) with treatment. The assay drug rangeis from about 20 nm to about 0.0001 nM for treatments Ex-vivo with NBconstructs of the invention.

Cell samples from patients diagnosed with other indications may also beobtained and similarly tested.

6.2. General Ex-Vivo Sensitivity Assay

Tumor samples are tested outside of a patient in a laboratory setting asa method to help predict which patients may respond to one or more NBagents.

Primary patient tumor material from blood or tissue is treated in asimilar manner to the above assays, depending on the tumor type. Tumoris removed via total blood collection or tissue biopsy and plated intoan appropriate tissue culture plate for 0-24 hours. The tumor specimenis then treated with varying concentrations of NB agent(s) over adilution series (e.g., starting concentration could be 20 nM diluted 3-4fold over 10 dilutions). The tissue culture plate is then treated withNB agent(s) for 24-72 hrs or for several hours (1-24) at 37° C. in theCO₂ supplied incubator. After incubation, the plated specimen isanalyzed using a method to detect the response of the specimen totreatment. Such as in the above examples; Cell Titer Glo (Promega#G7571) for cell viability, or Caspase 3/7 activation, or AnnexinVstaining, or Cell counting, or FACS analysis or other method todetermine the growth, death, or response to NB agent treatment areprovided.

Either a NB agent-sensitive cell line (e.g., Jurkat) or a NBagent-insensitive cell line (BE13), or PBMC (peripheral bloodmononuclear cells, e.g., made fresh from the blood of a healthyvolunteer) or the combination of the cell culture (cell line) spikedinto the PBMC are treated with various concentrations of NB agent(s) for3.5 hours at 37° C. in the CO₂ supplied incubator, after which Caspase3/7 activation is assessed (Promega #G7790) by following the directionssupplied by the manufacturer.

7. Example VII 7.1. Analysis of the RAF-MEK-ERK Pathway and DR5 Activity

Death Receptor 5 (DR5) is of particular interest in cancer drugdiscovery, since its activation selectively induces apoptosis in cancercells while sparing many normal cells. A DR5 specific agonist constructmay avoid the limitations of the ligand, namely binding to DecoyReceptors such as DcR1, DcR2 and OPG. A major hurdle for the clinicaldevelopments of DR5 agonist antibodies has been the difficulty inidentifying biomarkers predictive of efficacy in patient subgroups. Theanalysis of data from 200 cancer cell lines indicated that no obviousgenetic mutations, including those in RAS and RAF, are correlative ofsensitivity to LBY135, a DR5 agonist antibody (Novartis AG).

A strategy to screen pooled shRNAs based on DR5 sensitization/rescue isused to identify genes or pathways that modify DR5 mediated apoptosisafter genetic perturbation. The screens are conducted in seven cancercell lines across various lineages, and Solexa based deep sequencing ofthe integrated hairpins is used to de-convolute the shRNA compositions.

A set of common sensitizers and rescuers are identified as known pathwaycomponents, such that shRNAs targeting caspase 8, caspase 3 and DR5itself are the top rescuers and those targeting BCLxl are the topsensitizers, indicating the robustness of the screens. Interestingly, inaddition to these sets of common genes, inhibition of BRAF-MEK-ERKexhibits opposing cell-context-dependent phenotypes on DR5 mediatedapoptosis. Specifically, inhibition of BRAF-MEK-ERK pathway sensitizesMiapaca2 cells to DR5 mediated apoptosis, but rescues Colo205 cells fromit.

These opposite phenotypes correlate well with different kinetics ofcaspase 8 and caspase 9 activity, but appear to be independent of DR5expression level. Pathway dissection reveals that cFLIP and cIAP1, twoof the endogenous apoptosis inhibitors, mediate cross-talk between DR5mediated apoptosis and the BRAF-MEK-ERK pathway. In Miapaca2 cells,combination of U0126 (MEK inhibitor) and DR5 antibody accelerate thedegradation of cFLIP and cIAP1 proteins, while in colo205, both cFLIPand cIAP1 mRNAs are up-regulated. Consistently, inhibition of cFLIP orcIAP1 by shRNAs or chemical inhibition eliminate the rescue phenotype inColo205 cells, and further sensitize Miapaca2 to DR5 mediated apoptosis.Microarray analysis of Miapaca2 and Colo205 cells treated with MEKinhibitor reveal genes that are down-regulated in both cell lines,including DUSP6, ETV5 and ERG1, which are the canonical downstreamtargets of ERK.

7.2. Bcl-xL, cIAP1 and BRAF-MEK-ERK as Cell-Context Dependent Regulatorsof DR5 Mediated Apoptosis

Many proteins work as cell-context dependent regulators, if it isdefined broadly. For examples, ABT737, a Bcl-xL inhibitor, showed arange of phenotypes on LBY135 induced apoptosis. Among 35 cell lines tobe examined, it strongly sensitizes the effect of LBY135 on 7 celllines, mildly sensitizes 9 cell lines, but had no effect on the other 19cell lines. One of the plausible reasons is that parallel pathways arepresent in some cell lines that compensate the inhibition of Bcl-xL. Forexample, analysis of our screens reveal that shRNAs against Bcl-xLsensitize all cell lines except for Miapaca2 cells, which actually aresensitized by shRNAs targeting MCL1. Such kind of regulators would nothinder the development of combination therapy. In this aspect, aninhibitor to cIAP1 is a similar regulator that sensitizes a DR5 agonistantibody in a large number of cell lines (Table 26, plus data notshown). Interesting, cIAP1, not x-IAP, is specifically involved in thecross-talk between DR5 and BRAF-MEK-ERK pathway.

The BRAF-MEK-ERK pathway falls into a category forcell-context-dependent regulators. Its cell-context dependent effectsranged from sensitizer to antagonizer. Such dramatic differences providean opportunity to gain insight in the signaling networks regulating DR5mediated apoptosis. However, biomarker should be identified, or suchcombination will be difficult to develop, even if the sensitizingeffects are strong and desirable. Actually for DR5 resistant cell lines,such as ES2 and A375, BRAF/MEK inhibitors are the only partners in thetable below that show strong sensitizing effects, whereas inhibitors toBcl-xL do not. Downregulation of cIAP1 is associated with thesensitizing effects in multiple cell lines, suggesting it as a PDbiomarker for such combination strategy. Our results suggest that atriple combination of a DR5 agonist antibody with inhibitors to c1 andBRAF-MEK could be a solution.

Table 26 shows the synergistic effect between DR5 agonist antibody andinhibitors to cIAP1 or Bcl-xL. Thirty five cell lines are treated withLBY135 (DR5 agonist antibody, Novartis AG) alone or in combination withLBW242 (cIAP1 inhibitor) or ABT737 (Bcl-xL inhibitor). Cells are labeledas insensitive where the viability is higher than 70% at 5 nM of LBY135.For combination effects, it is labeled as ‘+++’ where the combinationreduces cell viability by more than 30%; and labeled as ‘++’ where thecombination reduces cell viability by between 10-30%. It is labeled as“none” when no synergy is observed with the combination treatment.

TABLE 26 Synergistic effect between DR5 agonist and clAP1 or Bcl-xLinhibitors. Sensitivity Sensitized Sensitized to LBY135 by by Cell lineTumor type (5 nM) LBW242 ABT737 A172 CNS sensitive ++ ++ Bxpc3Pancreatic insensitive ++ ++ Caov3 ovary sensitive ++ ++ H596 NSCLCinsensitive ++ ++ panc10.05 Pancreatic insensitive ++ +++ A375 Melanomainsensitive ++ none DU145 Prostate insensitive ++ none SK-Mel5 Melanomainsensitive ++ none colo741 Colon insensitive +++ ++ HCT116 Colonsensitive +++ ++ HT29 Colon insensitive +++ ++ colo205 Colon sensitive+++ +++ EBC1 Lung sensitive +++ +++ colo201 Colon sensitive +++ none ES2ovary insensitive +++ none sw626 Colon insensitive none ++ H2452Mesothelioma sensitive none +++ HPAC Pancreatic sensitive none +++ LNcapProstate insensitive none +++ T24 Bladder insensitive none +++ A549NSCLC insensitive none none Calu6 Lung sensitive none none H146 SCLCinsensitive none none H358 NSCLC sensitive none none H520 NSCLCinsensitive none none MDAMB361 Breast insensitive none none MDA-MB-453Breast sensitive none none OPM2 Multiple sensitive none none Myelomapanc2.03 Pancreatic insensitive none none Panc3.27 Pancreatic sensitivenone none RKO Colon insensitive none none RT4 Bladder insensitive nonenone SKOV3 ovary insensitive none none SW480 Colon sensitive none noneU266B1 Multiple insensitive none none Myeloma ++: sensitizing; +++:strong sensitizing;

In addition, 180 genes are also identified as being differentiallyregulated by MEK inhibition between the two cell lines. A set oftranscription factors that could be responsible for regulating thosegenes are predicted by gene set enrichment analysis (GSEA), followed byindividual examination using shRNA to confirm their involvement. Amongmany transcription factors, only FOXO3 and SP1 are found to be directlyinvolved in the cross-talk between the two pathways, as shown by theirrole in the regulation of cFLIP or cIAP1 at the mRNA level in Colo205cells. However, in Miapcaca2 cells, cFLIP or cIAP1 are regulated at theprotein level in proteasome and caspase 8 dependent manners. Finally,reduced cIAP1 protein levels are associated with the sensitizing effectsin additional cell lines. These results identify multiple potentcombination strategies for the DR5 agonist antibody LBY135, and reveal acell-context-dependent regulation of DR5 mediated apoptosis by theBRAF-MEK-ERK pathway.

To validate the shRNA results, chemical inhibitors specific to BRAF andMEK are used to disrupt the BRAF-MEK-ERK pathway. Namely, RAF265 andU0126 are inhibitors of BRAF and MEK, respectively. Inhibition of theBRAF-MEK-ERK pathway with either compound sensitized Miapaca2 cells toLBY135 treatment, but rescued Colo205 cells from it. Other BRAF and MEKspecific inhibitors gave similar phenotypes.

Miapaca2 cells have an activating KRAS mutation (G12C), whereas Colo205cells have an active BRAF mutation (V600E). So it is possible that thesemutations are responsible for the different phenotypes. However, H2122,which has the same KRAS mutation as Miapaca2 cells, did not show anysensitizing effect as in Miapaca2. Furthermore, after screening sevenadditional cell lines, all with BRAF mutations, we found that MEKinhibitors showed no effect in some cell lines while the others aresensitized as in Miapaca2 cells. Taken together, our results indicatethat neither BRAF nor KRAS mutations explain the cell-context dependentroles of the BRAF-MEK-ERK pathway on DR5 mediated apoptosis.

Overall, inhibition of BRAF-MEK-ERK is a double-edge sword—it can leadto either a sensitizing or an antagonizing effect when used incombination with a DR5 agonist. Inhibitors of cIAP1 or XIAP, of Bcl-xLor of cFLIP in combination with a DR5 agonist is sufficient forsensitization. However, the combination of a BRAF inhibitor and either aMEK or ERK inhibitor appear to antagonize the effect of pre-treatmentwith LBY135. While this raises concerns for combination therapytargeting both pathways in the clinic, data suggests that a triplecombination with cIAP inhibitors could be a solution for the antagonism.Namely, a triple combination of a DR5 agonist, a cIAP inhibitor (eitherto cIAP1 or to XIAP) and the choice of a MEK inhibitor or a BRAFinhibitor should also result in synergistic induction of cell death.

7.3. Experimental Protocols

For the shRNA library screen, target cells are infected with a pooledshRNA library targeting kinome and apotome shRNA, selected withpuromycin, then treated with cross-linked LBY135 or PBS control. Samplescollected at Day 0, 7 and 14 are subjected to DNA extraction, PCRamplification of integrated shRNAs and purification, followed by solexasequencing. Comparisons between treatments at the same time pointsreveal shRNAs modulating DR5 mediated apoptosis. Comparisons betweentime points in PBS controls reveal effects of shRNAs on cell growth.These hits are then subjected to individual validation.

To analyze knock-down of LIMK2 sensitized Miapaca2 and Colo205 to LBY135induced apoptosis, cells are infected with shRNAs against LIMK2 orcontrol hairpins, selected with puromycin for 5 days, then seeded in96-well-plates for treatments with serial dilutions of cross-linkedLBY135 the next day for 36 hours. Cell viabilities are measured bycell-titer-glow reagent, and normalized to the samples not treated withLBY135. Cell survival rate is plotted as a function of concentrations togenerate a dose-response curve. Data includes the mean±SD of triplicatesof a representative experiment out of at least two independentexperiments.

Chemical inhibitors of MEK and BRAF are used to validate the phenotypeof shRNAs in the screen. Miapaca2 and Colo205 are pre-incubated withDMSO, MEK inhibitor U0126 (10 uM) or BRAF inhibitor RAF265 (1 uM)overnight, then treated with serial dilutions of cross-linked LBY135 for36 hours. Cell viabilities are measured by cell-titer-glow reagent, andnormalized to the samples not treated with LBY135. Cell survival rate isplotted as a function of concentrations to generate a dose-responsecurve. Data are the mean±SD of triplicates of a representativeexperiment out of at least two independent experiments.

To show that activation of ERK by DR5 remains intact in both cell lines,Miapaca2 and Colo205 cells are pre-incubated with DMSO or U0126 (10 uM)overnight, treated with cross-linked LBY135 (0.25 nM), and collected at0 h, 1 h, 3 h and 6 h for western blot analysis for ERK and p-ERK. MEKinhibition decreases DR5 induced activities of caspase-8, caspase-9 andcaspase-3/7 in Colo205 cells, whereas it increases them in Miapaca2cells. Colo205 and Miapaca2 are treated with cross-linked LBY-135 at 0nM, 0.25 nM or 1 nM for 7 hrs. Cells are lysed for analysis ofactivities of caspase8, caspase9 and caspase3/7. Caspase activities areplotted as a function of LBY135 concentrations. Data is representativeexperiment out of at least two independent experiments.

Microarray analysis is used to reveal those genes with significantcorrelation between Colo205 and Miapaca2 cells among genes regulated byMEK. Colo205 and Miapaca2 cells are pre-incubated with DMSO or U0126overnight in five independent experiments. RNA is extracted formicroarray analysis. Relative expression level for each gene in U0126versus DMSO treated cells are plotted as a function of Colo205 cells onY-axis and Miapaca2 on X-axis.

7.4. Working Model for Cross-Talk Between DR5 and RAF-MEK-ERK

A working model proposes that treatment with a DR5 agonist activatesboth caspase-8 and ERK. Cross-talks between these two pathways aremediated by cFLIP and cIAP1, which negatively regulate the activation ofcaspase-8 and caspase-9 that in turn control the apoptosis induced byDR5 agonist. cFLIP and cIAP1 are regulated at different levels in amanner that is cell-context dependent. In Colo205 cells, they areregulated at their RNA levels through transcription factor FOXO3 andSP1, which themselves are regulated by BRAF-MEK-ERK pathway. In Miapaca2cells, cFLIP and cIAP1 are regulated by BRAF-MEK-ERK pathway at proteinlevel in proteasome and caspase 8 dependent manners.

8. Example VIII 8.1. Exemplary Biacore Assay

A suitable assay for determining whether an amino acid sequence or otherbinding agent cross-blocks or is capable of cross-blocking is a Biacoreassay. It will be appreciated that the assay can be used with any of theamino acid sequences (or other binding agents such as polypeptides ofthe invention) described herein.

The Biacore machine (for example the Biacore 3000) is operated in linewith the manufacturers recommendations. In one exemplary cross-blockingassay, the target protein is coupled to a CM5 Biacore chip usingstandard amine coupling chemistry to generate a surface that is coatedwith the target. Typically 200-800 resonance units of the target wouldbe coupled to the chip (i.e., an amount that gives easily measurablelevels of binding but that is readily saturable by the concentrations oftest reagent being used).

Two test amino acid sequences (termed A* and B*) to be assessed fortheir ability to cross-block each other are mixed at a one to one molarratio of binding sites in a suitable buffer to create the test mixture.When calculating the concentrations on a binding site basis themolecular weight of an amino acid sequence is assumed to be the totalmolecular weight of the amino acid sequence divided by the number oftarget binding sites on that amino acid sequence. The concentration ofeach amino acid sequence in the test mix should be high enough toreadily saturate the binding sites for that amino acid sequence on thetarget molecules captured on the Biacore chip. The amino acid sequencesin the mixture are at the same molar concentration (on a binding basis)and that concentration would typically be between 1.00 and 1.5micromolar (on a binding site basis). Separate solutions containing A*alone and B* alone are also prepared. A* and B* in these solutions arein the same buffer and at the same concentration as in the test mix.

The test mixture is passed over the target-coated Biacore chip and thetotal amount of binding recorded. The chip is then treated in such a wayas to remove the bound amino acid sequences without damaging thechip-bound target. Typically this is done by treating the chip with 30mM HCl for 60 seconds. The solution of A* alone is then passed over thetarget-coated surface and the amount of binding recorded. The chip isagain treated to remove all of the bound amino acid sequences withoutdamaging the chip-bound target. The solution of B* alone is then passedover the target-coated surface and the amount of binding recorded.

The maximum theoretical binding of the mixture of A* and B* is nextcalculated, and is the sum of the binding of each amino acid sequencewhen passed over the target surface alone. If the actual recordedbinding of the mixture is less than this theoretical maximum then thetwo amino acid sequences are cross-blocking each other. Thus, ingeneral, a cross-blocking amino acid sequence or other binding agentaccording to the invention is one which will bind to the target in theabove Biacore cross-blocking assay such that during the assay and in thepresence of a second amino acid sequence or other binding agent of theinvention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%)of the maximum theoretical binding, specifically between 75% and 0.1%(e.g. 75% to 4%) of the maximum theoretical binding, and morespecifically between 70% and 0.1% (e.g. 70% to 4%) of maximumtheoretical binding (as just defined above) of the two amino acidsequences or binding agents in combination.

The Biacore assay described above is a primary assay used to determineif amino acid sequences or other binding agents cross-block each otheraccording to the invention. On rare occasions particular amino acidsequences or other binding agents may not bind to target coupled viaamine chemistry to a CM5 Biacore chip (this usually occurs when therelevant binding site on target is masked or destroyed by the couplingto the chip). In such cases cross-blocking can be determined using atagged version of the target, e.g., a N-terminal His-tagged version (R &D Systems, Minneapolis, Minn., USA; 2005 cat#1406-ST-025). In thisparticular format, an anti-His amino acid sequence would be coupled tothe Biacore chip and then the His-tagged target would be passed over thesurface of the chip and captured by the anti-His amino acid sequence.The cross blocking analysis would be carried out essentially asdescribed above, except that after each chip regeneration cycle, newHis-tagged target would be loaded back onto the anti-His amino acidsequence coated surface. In addition to the example given usingN-terminal His-tagged DR5, C-terminal His-tagged target couldalternatively be used. Furthermore, various other tags and tag bindingprotein combinations that are known in the art could be used for such across-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tagwith anti-FLAG antibodies; biotin tag with streptavidin).

9. Example IX 9.1. Exemplary ELISA Assay

The following generally describes an ELISA assay for determining whetheran amino acid sequence or other binding agent directed against a targetcross-blocks or is capable of cross-blocking as defined herein. Thisassay can be used with any of the amino acid sequences (or other bindingagents such as polypeptides of the invention) herein.

The general principal of the assay is to have an amino acid sequence orbinding agent that is directed against the target coated onto the wellsof an ELISA plate. An excess amount of a second, potentiallycross-blocking, anti-target amino acid sequence is added in solution(i.e., not bound to the ELISA plate). A limited amount of the target isthen added to the wells. The coated amino acid sequence and the aminoacid sequence in solution compete for binding of the limited number oftarget molecules. The plate is washed to remove excess target that hasnot been bound by the coated amino acid sequence and to also remove thesecond, solution phase amino acid sequence as well as any complexesformed between the second, solution phase amino acid sequence andtarget. The amount of bound target is then measured using a reagent thatis appropriate to detect the target. An amino acid sequence in solutionthat is able to cross-block the coated amino acid sequence will be ableto cause a decrease in the number of target molecules that the coatedamino acid sequence can bind relative to the number of target moleculesthat the coated amino acid sequence can bind in the absence of thesecond, solution phase, amino acid sequence.

In the instance where the first amino acid sequence, e.g. an Ab-X, ischosen to be the immobilized amino acid sequence, it is coated onto thewells of the ELISA plate, after which the plates are blocked with asuitable blocking solution to minimize non-specific binding of reagentsthat are subsequently added. An excess amount of the second amino acidsequence, i.e., Ab-Y, is then added to the ELISA plate such that themoles of Ab-Y [target] binding sites per well are at least 10 foldhigher than the moles of Ab-X [target] binding sites that are used, perwell, during the coating of the ELISA plate. [target] is then added suchthat the moles of [target] added per well are at least 25-fold lowerthan the moles of Ab-X [target] binding sites that are used for coatingeach well. Following a suitable incubation period the ELISA plate iswashed and a reagent for detecting the target is added to measure theamount of target specifically bound by the coated anti-[target] aminoacid sequence (in this case Ab-X).

The background signal for the assay is defined as the signal obtained inwells with the coated amino acid sequence (in this case Ab-X), secondsolution phase amino acid sequence (in this case Ab-Y), [target] bufferonly (i.e., no target) and target detection reagents. The positivecontrol signal for the assay is defined as the signal obtained in wellswith the coated amino acid sequence (in this case Ab-X), second solutionphase amino acid sequence buffer only (i.e., no second solution phaseamino acid sequence), target and target detection reagents. The ELISAassay may be run in a manner so as to have the positive control signalbe at least six times the background signal.

To avoid any artifacts (e.g. significantly different affinities betweenAb-X and Ab-Y for [target]) resulting from the choice of which aminoacid sequence to use as the coating amino acid sequence and which to useas the second (competitor) amino acid sequence, the cross-blocking assaymay to be run in two formats: (1) format 1 is where Ab-X is the aminoacid sequence that is coated onto the ELISA plate and Ab-Y is thecompetitor amino acid sequence that is in solution and (2) format 2 iswhere Ab-Y is the amino acid sequence that is coated onto the ELISAplate and Ab-X is the competitor amino acid sequence that is insolution. Ab-X and Ab-Y are defined as cross-blocking if, either informat 1 or in format 2, the solution phase anti-target amino acidsequence is able to cause a reduction of between 60% and 100%,specifically between 70% and 100%, and more specifically between 80% and100%, of the target detection signal {i.e., the amount of target boundby the coated amino acid sequence) as compared to the target detectionsignal obtained in the absence of the solution phase anti-target aminoacid sequence (i.e., the positive control wells).

10. Example X 10.1. Macrophage Depletion Experiment

All anti-DR5 antibodies in clinical development to date requirecross-linking to achieve optimal potency in vitro and in vivo. In vivo,this crosslinking is believed to be mediated via the binding of the Fcportion of the anti-DR5 antibody to Fc receptor expressing immune cells.To investigate the cross-linking effects in vivo of the anti-DR5antibody LCR211 compared with an NB construct, which is expected to notrequire this crosslinking, the activity of the mouse IgG1 anti-DR5antibody LCR211 is compared to 11H6 tetra under tumor associatedmacrophage (TAM) depletion conditions in the MiaPaCa-2 xenograft modelin the NOD/LtSz-scid IL2R gamme^(null) female mice (NSG mouse)background. NSG (NOD-SCID gamma) mice are deficient in T cells, B cells,and have little or no NK cell cytotoxicity activity caused by disruptedcytokine signaling as a result of the deletion of the IL-2R gamma-chain(Shultz, J. of Immunology 2005). Depletion of TAMs is accomplished usinga small molecule that inhibits signaling through the CSF-1 receptor.Three doses at 200 mg/kg once per day depletes up to 80% of macrophages,as previously shown. Daily dosing is continued throughout the study.

MiaPaCa cells are harvested in exponential growth. Five million cellsmixed 50:50 with Matrigel are subcutaneously implanted into the upperright flank of nude mice. For cell implantation, mice are anesthetizedwith continuous flow of 2-4% isoflurane/20 oxygen mixture using theintegrated multi chambers anesthesia center (IMCAC) and inductionchamber (Vetequip. Inc., Pleasanton, Calif.). The tumor take rateis >90% and tumors reach approximately 150-350 mm³ around 14 days postcell implantation. Animals are randomized according to tumor volume suchthat the mean tumor volume and range are statistically similar betweentreatment groups (as determined by a Student's t-test). Tumors aremeasured with digital calipers twice a week at the start of dosing.Tumor volumes are calculated using the ellipsoid formula:(length×width²)/2. LCR211 is dosed at 10 mg/kg 3qw iv and 11H6 tetra isdosed at 10 mg/kg and 40 mg/kg qw iv, with or without the CSF1-Rinhibitor (CSF-1Ri) at 250 mg/kg qd po.

Percent treatment/control (T/C) values are calculated using thefollowing formula:

% T/C=100×ΔT/ΔC if ΔT>0%

Regression=100×ΔT/T _(initial) if ΔT<0

where:

-   -   T=mean tumor volume of the drug-treated group on the final day        of the study;    -   ΔT=mean tumor volume of the drug-treated group on the final day        of the study−mean tumor volume of the drug-treated group on        initial day of dosing;    -   T_(initial)=mean tumor volume of the drug-treated group on        initial day of dosing;    -   C=mean tumor volume of the control group on the final day of the        study; and    -   ΔC=mean tumor volume of the control group on the final day of        the study−mean tumor volume of the control group on initial day        of dosing.    -   % T/C calculations are done at the end of study.

As seen in Table 27 and in FIGS. 4A and 4B, 11H6 tetra maintains potentsingle agent activity in the MiaPaCa-2 xenograft under TAM depletionconditions in the NSG mouse background, while under the same conditionsthe murine anti-DR5 antibody LCR211 loses efficacy.

TABLE 27 Summary of 11H6 tetra efficacy under TAM depletion conditions %T/C no % T/C with Molecule TAM depletion TAM depletion LCR211 10 mg/kg3qw     22%    95% 11H6 tetra 10 mg/kg qw −23.64% −5.73% 11H6 tetra 40mg/kg qw −67.77% −9.11%

10.2. Anti-Tumor Activity of a DR5 Agent in LCR211-InsensitivePatient-Derived Tumor Model

TPAN1-IFA, a patient-derived pancreatic tumor (Xentech), is implantedsubcutaneously (sc) into (80) female nude mice (Harlan, about 8 weeks ofage at implant). Animals are randomized at start of treatment intogroups of nine. Starting tumor volume is 99 mm³ (range 63-196 mm³).Treatment groups are: (1) Vehicle, once a week (qw), intravenously(i.v.), (2) LCR211, 10 mg/kg, three times a week (3qw), i.v., (3) 11H6tetramer, 10 mg/kg, qw, i.v., (4) 11H6, 40 mg/kg, qw, i.v., 5.Gemcitabine, 60 mg/kg, twice a week (2qw), i.v. Intravenous dosingvolume is 5 ml/kg. Mice are weighted twice a week. Tumors are calliperedtwice a week and tumor volume (TV) calculated using the formula: TV(mm³)=[length (mm)×width (mm)2]/2 is used, where the length and thewidth are the longest and the shortest diameters of each tumor,respectively.

As shown in FIG. 5, after 4 weeks of dosing, LCR211 shows no singleagent activity, with the mean change in tumor volume of the treatmentgroup over control (T/C)=109%. In contrast, 11H6 dosed at 10 mg/kgweekly and at 40 mg/kg weekly results in 1% and 86% regression,respectively (p<0.05 vs vehicle). Gemcitabine treatment results in a T/Cof 25% (p<0.05 vs vehicle). LCR211 and 11H6 treatment is well toleratedwith no significant loss of body weight observed. Thus, in apatient-derived cancer that is insensitive to conventional antibodytargeting of DR5, the NB constructs show potent tumor regression. Inthis example, the NB construct has the potential to treat a larger classof patients that could be refractory to conventional targetingapproaches for this pathway.

11. Example XI 11.1. X-Ray Crystallographic Structure Determination ofthe Human DR5/NB Construct Complexes

Crystal structures of a human DR5 ECD fragment bound to monomericconstructs 11H6 (SEQ ID NO: 103) and 4E6 (SEQ ID NO: 104) aredetermined. As detailed below, individual DR5 protein fragments areexpressed, purified and mixed to form complexes. Protein crystallographyis employed to generate atomic resolution data for the DR5 protein boundto the two examples.

Two variants of DR5 protein are produced for crystallography, namelyDR5_(—)54 and DR5_(—)61. Following the sequence numbering of human DR5,GenBank accession number BAA33723.1 (SEQ ID NO: 89), the DR5 sequencescorrespond to residues 54 to 183, and 61 to 183, respectively. As shownin Table 28, sequences in lower case letters are removed duringproduction. For DR5_(—)54 (SEQ ID NO: 100) and DR5_(—)61 (SEQ ID NO:101), human DR5 sequence is underlined. In Table 28, “ID” represents theSEQ ID NO.

TABLE 28  Proteins used for crystal structure determination ConstructAmino acid sequence in one letter code. ID HuDR5meqrgqnapaasgarkrhgpgpreargarpglrvpktlvlvvaavlllvsaesali 89 GenBank:tqqdlapqqrvapqqkrsspseglcppghhisedgrdcisckyggdysthwndllf BAA33723.1clrctrcdsgevelspctttrntvcqceegtfreedspemcrkcrtgcprgmvkvgdctpwsdiecvhkesgtkhsgeapaveetvtsspgtpaspcslsgiiigvtvaavvlivavfvcksllwkkvlpylkgicsggggdpervdrssqrpgaednvlneivsilqptqvpeqemevqepaeptgvnmlspgesehllepaeaersqrrrllvpanegdptetlrqcfddfadlvpfdsweplmrklglmdneikvakaeaaghrdtlytmlikwvnktgrdasvhtlldaletlgerlakqkiedhllssgkfmylegnadsams DR5_54mvsaivlyvllaaaahsafaadlgslevlfgALITQQDLAPQQRAAPQQKRSSPSE 100 gp67DR5a54-GLCPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRN s183-PHTVCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESAAALEVLFQgpssgklghhhhhhhhhh DR5_61mvsaivlyvllaaaahsafaadlgslevlfqGPSMALAPQQRAAPQQKRSSPSEGL 101 gp67DR5l61-CPPGHHISEDGRDCISCKYGQDYSTHWNDLLFCLRCTRCDSGEVELSPCTTTRNTV s183-PHCQCEEGTFREEDSPEMCRKCRTGCPRGMVKVGDCTPWSDIECVHKESAAALEVLFQgpssgklghhhhhhhhhh 11H6 humkktaiaiavalaglatvaqaEVQLLESGGGLVQPGGSLRLSCAASGTFDKINNMG 102 pAX100-11H6WYRQAPGKQRDLVAQITPGGITDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCNAEILKRAYIDVYVNYWGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH 4E6 humkktaiaiavalaglatvaqaEVQLLESGGGLVQPGGSLRLSCAASGRTFGSIRVG 103 pAX111-4E6WFRQAPGKGREFVSAINRNDGTTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAGLQYNRAADRVPVGAVYWGQGTLVTVSSHHHHHH

The above proteins are expressed in SF9 cells using a baculovirusexpression system, with the expression vector containing the GP67 signalpeptide. DR5 protein is purified from the cell growth media following a2.5 day infection. The media is clarified with the addition of 5 mMCaCl₂, 1 mM NiCl₂, 50 mM Tris pH 8.0, and 1 μM PMSF. The protein iscaptured on Ni-NTA resin (Qiagen) equilibrated in 50 mM Tris pH 8.0, 300mM NaCl using a gravity flow column. The column is washed with 50 mMTris pH 8.0, 300 mM NaCl, 30 mM imidazole, followed by elution with 50mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. Eluted DR5 from the Nicolumn is cleaved with PreScission protease (GE Healthcare), followed bygel filtration chromatography using a Superdex 75 column (GE Healthcare)equilibrated in 50 mM HEPES pH 7.6, 150 mM NaCl. DR5 is further purifiedby ion exchange using a MonoQ column (GE Healthcare) at pH 7.5 with a 20column volume gradient of 0.03-1 M NaCl. Peak fractions are analyzed bySDS-PAGE and LCMS prior to pooling.

11.2. 4E6 and 11H6 Crystallization with DR5

Both 4E6 (pAX111-4E6, SEQ ID NO:103) and 11H6 (pAX100-11H6, SEQ IDNO:102) are cloned as monomeric NB constructs for expression in E coliusing BL21(DE3)pLysS cells and BL21(DE3)Star cells, respectively, andboth contain signal sequences for periplasmic localization, as providedin Table 28 above. Following a 3 hour induction with IPTG at 37° C.,cells are harvested and lysed. Protein is captured on a Ni-NTA columnpre-equilibrated in 50 mM Tris pH 8.0, 300 mM NaCl. The column is washedwith 50 mM Tris pH 8.0, 300 mM NaCl, 50 mM imidazole, followed byelution with 50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. The Nieluate is further purified via gel filtration chromatography using aSuperdex 75 column (GE Healthcare). Whereas this is the finalpurification step for 11H6, the 4E6 underwent an additional purificationstep via a MonoS cation exchange column (GE Healthcare).

A complex of DR5_(—)61/4E6 is prepared by mixing DR5_(—)61 and 4E6 in a1:1 molar ratio (concentration measured via LCUV), incubating on ice for1 hour, and purifying the complex on a Superdex75 column (GE Healthcare)equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl. Peak fractions areanalyzed by SDS-PAGE and LCMS. Fractions containing DR5_(—)61/4E6 areconcentrated to about 10 mg/ml for crystallization.

A complex of DR5_(—)54/11H6 is prepared by mixing DR5_(—)54 and 11H6 ina molar ration of 1:1.3 (concentration estimated by LCUV), incubating onice for 1 hour, and purifying the complex on a Superdex75 column (GEHealthcare) equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl. Peakfractions containing the DR5_(—)54/11H6 dimer are pooled andconcentrated to about 12.7 mg/ml. The dimer is then Trypsin-treated asfollows: 200 μl of dimer (about 2.5 mg) is used to resuspend 20 μg oflyophylized trypsin, which is then incubated at room temperature for 15minutes. The DR5_(—)54/11H6/trypsin mixture is centrifuged prior tosetting up crystallization screens.

Crystals are grown by sitting drop vapor diffusion from drops containingequal volumes of protein and reservoir solution. For DR5_(—)61/4E6complex a reservoir solution of 22% PEG 3350, 150 mM calcium acetate,100 mM HEPES pH 7. 5 produced crystals upon incubation at 20° C. Fortrypsin-treated DR5_(—)54/11H6 complex a reservoir solution of 0.2 Mammonium sulfate, 0.1M Tris pH 8.5, 25% PEG 3350 produced crystals uponincubation at 20° C.

The DR5_(—)61/4E6, crystals are transferred to a cryo solutioncontaining 25% PEG 3350, 50 mM HEPES pH 7.5, 150 mM calcium acetate, 15%glycerol, 10% ethylene glycol and flash cooled in liquid nitrogen. TheDR5_a54/11H6, crystals are transferred to reservoir solution containingadditional 22% glycerol and flash cooled in liquid nitrogen.

For DR5_(—)61/4E6 complex, diffraction data are collected at stationPXI-X06SA at the Swiss Light Source (Paul Scherrer Institut, Villigen,Switzerland). Data are processed and scaled at 1.9 Å using autoPROC(Global Phasing, LTD) in space group C121 with cell dimensions a=98.50Å, b=84.65 Å, c=64.91 Å, alpha=90°, beta=99.38°, gamma=90°. TheDR5_(—)61/4E6 structure is solved by molecular replacement using Phaser(McCoy et al., (2007) J. Appl. Cryst. 40: 658-674) with DR5 structure2H9G and the camelized human VH structure 1OL0 as search models. Thefinal model, which contains 2 molecule of the DR5_(—)61/4E6 complex perasymmetric unit, is built in COOT (Emsley & Cowtan (2004) Acta Cryst.60: 2126-2132) and refined to R and R_(free) values of 22.2% and 24.1%,respectively, with an rmsd of 0.003 Å and 0.69° for bond lengths andbond angles, respectively, using PHENIX (Adams et al., Acta Cryst. D66,213-221 (2010)).

For DR5_(—)54/11H6 complex, diffraction data are collected at beamline17-ID at the Advanced Photon Source (Argonne National Laboratory, USA).Data are processed and scaled at 2.2 Å using autoPROC (Global Phasing,LTD) in space group P6₅ with cell dimensions a=99.61 Å, b=99.61 Å,c=107.73 Å, alpha=90°, beta=90°, gamma=120°. The DR5_(—)54/11H6structure is solved by molecular replacement using Phaser (McCoy et al.,(2007) J. Appl. Cryst. 40: 658-674) with DR5 structure 2H9G and NBconstruct 4E6 as search models. The final model, which contains 2molecule of the DR5_(—)54/11H6 complex per asymmetric unit, is built inCOOT (Emsley & Cowtan (2004) Acta Cryst. 60: 2126-2132) and refined to Rand R_(free) values of 19.5% and 22.0%, respectively, with an rmsd of0.002 Å and 0.64° for bond lengths and bond angles, respectively, usingPHENIX (Adams et al., Acta Cryst. D66, 213-221 (2010)).

11.3. 4E6 and 11H6 Recognize Unique Epitopes on DR5

The crystal structure of the 4E6/DR5_(—)61 complex has been used toidentify the DR5 epitope for the 4E6 construct. The interaction surfaceon DR5 is formed by three discontinuous (i.e., noncontiguous) sequences:namely residues 77 through 80, residues 87 through 91, and residues 105through 114, as detailed in Table 29A. These residues form thethree-dimensional surface that is recognized by the NB construct.Interactions include backbone interactions, solvent mediatedinteractions, and direct sidechain interactions. The amino acids whosesidechains are directly contributing to the interactions noted in Table29A. The interaction surface contribution from the 4E6 NB construct isformed by its N-terminal residue 2, and three loop regions: residues 28through 33, residues 53 through 59, and residues 99 through 115. Thecontact residues are listed in Table 29B.

In Table 29A, DR5 residues that contain atoms in contact with the NBconstruct, 4E6 are listed. Contact is defined to be within 5 Angstromsof the NB construct to account for potential water mediatedinteractions. Amino acids whose sidechains contribute directly to theinteraction surface are noted with a “+”.

TABLE 29A DR5 Conformational Epitope for 4E6 Sequence Sidechain ProteinAmino acid position Interaction DR5 S 77 DR5 E 78 + DR5 G 79 DR5 L 80 +DR5 I 87 + DR5 S 88 DR5 E 89 DR5 D 90 DR5 G 91 + DR5 T 105 DR5 H 106 +DR5 W 107 + DR5 N 108 DR5 D 109 + DR5 L 110 + DR5 L 111 + DR5 F 112 +DR5 L 114 +

In Table 29B, residues in the NB construct 4E6 that are in contact withDR5 are listed. Contact is defined to be within 5 Angstroms of the NBconstruct to account for potential water mediated interactions. Aminoacids whose sidechains contribute directly to the interaction surfaceare noted with a “+”.

TABLE 29B 4E6 amino acids in contact with DR5 Sequence Sidechain ProteinAmino acid position Interaction 4E6 V 2 + 4E6 T 28 + 4E6 G 30 + 4E6 S31 + 4E6 I 32 + 4E6 R 33 + 4E6 R 53 4E6 N 54 + 4E6 Y 59 4E6 G 99 + 4E6 L100 4E6 Q 101 + 4E6 Y 102 + 4E6 N 103 4E6 R 104 4E6 A 105 + 4E6 A 106 +4E6 V 111 + 4E6 V 114 + 4E6 Y 115 +

The crystal structure of the 11H6/DR5_(—)54 complex is used to identifythe DR5 epitope for the 11H6 construct. The interaction surface on DR5is formed by two discontinuous sequences, residues 86 through 102 andresidues 111 through 118, and two additional residues 74 and 125 asdetailed in Table 30A. These residues form the three-dimensional surfacethat is recognized by the NB construct. Interactions include backboneinteractions, solvent mediated interactions, and direct sidechaininteractions. The amino acids whose sidechains are directly contributingto the interactions noted in Table 30A. The interaction surfacecontribution from the 11H6 construct extends beyond the loop regionsobserved for the 4E6 construct. Residues 30 through 37, residues 44through 61 and residues 96 through 112 contribute to the binding surfaceof the 11H6 construct as detailed in Table 30B. The 11H6 construct'sloop from residues 98 to 110 is in a very different conformation thanthe corresponding loop from the 4E6 construct. This alternate loopposition provides a unique interaction surface for DR5 binding.

In Table 30A, DR5 residues that contain atoms in contact with the 11H6construct are listed. Contact is defined to be within 5 Angstroms of11H6 to account for potential water mediated interactions. Amino acidswhose sidechains contribute directly to the interaction surface arenoted with a “+”. Buried surface area is determined as a percentage.

TABLE 30A DR5 Conformational Epitope for 11H6 Sequence Buried surfaceProtein Amino acid position area (%) DR5 S 74 DR5 H 86 + DR5 I 87 DR5 S88 + DR5 E 89 + DR5 D 90 + DR5 R 92 DR5 D 93 + DR5 I 95 + DR5 S 96 DR5 K98 + DR5 G 100 DR5 Q 101 + DR5 D 102 + DR5 L 111 + DR5 F 112 + DR5 C 113DR5 R 115 + DR5 R 118 + DR5 E 125

In Table 30B, residues in 11H6 that are in contact with DR5 are listed.Contact is defined to be within 5 Angstroms of the 11H6 construct toaccount for potential water mediated interactions. Amino acids whosesidechains contribute directly to the interaction surface are noted witha “+”.

TABLE 30B 11H6 amino acids in contact with DR5 Sequence SidechainProtein Amino acid position Interaction 11H6 K 30 + 11H6 N 32 + 11H6 N33 + 11H6 Y 37 + 11H6 Q 44 + 11H6 R 45 11H6 D 46 + 11H6 L 47 + 11H6 Q50 + 11H6 I 51 11H6 T 52 + 11H6 P 53 11H6 G 54 11H6 G 55 11H6 I 56 +11H6 T 57 11H6 D 58 + 11H6 A 60 + 11H6 D 61 11H6 N 96 + 11H6 E 98 + 11H6L 100 + 11H6 Y 104 11H6 D 106 11H6 V 107 + 11H6 Y 108 + 11H6 N 110 +11H6 W 112 +

EQUIVALENTS

One embodiment provides an isolated polypeptide comprising at least onemonomer of a single variable domain of a NB agent that specificallybinds to human DR5.

One embodiment provides the polypeptide of paragraph [00612], whereinsaid single variable domain is selected from the group consisting of a)singe variable domains comprising one or more complementaritydetermining region 3 (CDR3) sequences selected from any one or more ofSEQ ID NOs: 63-68; b) single variable domains comprising one or morecomplementarity determining region 3 (CDR3) sequences with 90% identityto at least one CDR3 selected from any one or more of SEQ ID NOs: 63-68;and c) single variable domains comprising one or more complementaritydetermining region 3 (CDR3) sequences with at least 95% identity to atleast one CDR3 selected from any one or more of SEQ ID NOs: 63-68.

One embodiment provides the polypeptide of paragraph [00612], whereinsaid single variable domain is selected from the group consisting of a)singe variable domains comprising one or more complementaritydetermining region 3 (CDR3) sequences selected from any one or more ofSEQ ID NOs: 41-44, 51-55 and 63-68; b) single variable domainscomprising one or more complementarity determining region 3 (CDR3)sequences with at least 90% identity to at least one CDR3 selected fromany one or more of SEQ ID NOs: 41-44, 51-55 and 63-68; and c) singlevariable domains comprising one or more complementarity determiningregion 3 (CDR3) sequences with at least 95% identity to at least oneCDR3 selected from any one or more of SEQ ID NOs: 41-44, 51-55 and 63-68with at least 90% identity.

One embodiment provides the polypeptide of paragraph [00612], whereinsaid single variable domain is selected from the group consisting of a)singe variable domains with SEQ ID NOs: 1-5, 26, 30 and 87; and b)single variable domains with at least 95% identity to at least onesingle variable domain with SEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides an isolated polypeptide comprising at leastthree monomers of a single variables domain that specifically binds tohuman DR5.

One embodiment provides the polypeptide of paragraph [00616], whereinsaid polypeptide comprises three identical monomers of the singlevariable domain and wherein said single variable domain is selected fromthe group consisting of a) a single variable domain comprising one ormore complementarity determining region 3 (CDR3) sequences selected fromany one or more of SEQ ID NOs: 63-68; b) single variable domainscomprising one or more complementarity determining region 3 (CDR3)sequences with at least 90% identity to at least one CDR3 selected fromany one or more of SEQ ID NOs: 63-68; and c) single variable domainscomprising one or more complementarity determining region 3 (CDR3)sequences with at least 95% identity to at least one CDR3 selected fromany one or more of SEQ ID NOs: 63-68 with at least 90% identity.

One embodiment provides the polypeptide of paragraph [00616], whereinsaid polypeptide comprises three identical monomers of the singlevariable domain and wherein said single variable domain is selected fromthe group consisting of a) singe variable domains comprising one or morecomplementarity determining region 3 (CDR3) sequences selected from anyone or more of SEQ ID NOs: 41-44, 51-55 and 63-68; b) single variabledomains comprising one or more complementarity determining region 3(CDR3) sequences with at least 90% identity to at least one CDR3selected from any one or more of SEQ ID NOs: 41-44, 51-55 and 63-68; andc) single variable domains comprising one or more complementaritydetermining region 3 (CDR3) sequences with at least 95% identity to atleast one CDR3 selected from any one or more of SEQ ID NOs: 41-44, 51-55and 63-68 with at least 90% identity.

One embodiment provides the polypeptide of paragraph [00616], whereinsaid polypeptide comprises three identical monomers of the singlevariable domain and wherein said single variable domain is selected fromthe group consisting of a) singe variable domains with SEQ ID NOs: 1-5,26, 30 and 87; b) single variable domains with at least 90% identity toat least one single variable domain with SEQ ID NOs: 1-5, 26, 30 and 87and c) single variable domains with at least 95% identity to at leastone single variable domain with SEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides the polypeptide of paragraph [00616] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequences with SEQ ID NO: 6, 9, 12-14, 17, 20, 27 and 31; and b)amino acid sequences with at least 90% identity to said amino acidsequence with SEQ ID NO: 6, 9, 12-14, 17, 20, 27 and 31.

One embodiment provides the polypeptide of paragraph [00616] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequences with SEQ ID NO: 6, 9, 12-14, 17, 20, 27 and 31; and b)amino acid sequences with at least 90% identity to said amino acidsequence with SEQ ID NO: 6, 9, 12-14, 17, 20, 27 and 31.

The polypeptide of paragraph 5 comprising an amino sequence that isselected from the group consisting of a) amino acid sequences with SEQID NO: 6, 9, 12-14, 17, 20, 27 and 31; and b) amino acid sequences withat least 95% identity to said amino acid sequence with SEQ ID NO: 6, 9,12-14, 17, 20, 27 and 31.

One embodiment provides an isolated polypeptide comprising at least fourmonomers of a single variable domain that specifically binds to humanDR5.

One embodiment provides the polypeptide of paragraph [00623], whereinsaid polypeptide comprises four single variable domains and wherein saidsingle variable domain is selected from one, two, three or four monomersfrom the group consisting of a) singe variable domains with SEQ ID NOs:1-5, 26, 30 and 87; b) single variable domains with at least 90%identity to said single variable domain with SEQ ID NOs: 1-5, 26, 30 and87; and c) single variable domains with 95% identity to said singlevariable domain with SEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides the polypeptide of paragraph [00623], whereinsaid polypeptide comprises four identical single variable domains andwherein said single variable domain is selected from the groupconsisting of a) singe variable domains with SEQ ID NOs: 1-5, 26, 30 and87; b) single variable domains with at least 90% identity to said singlevariable domain with SEQ ID NOs: 1-5, 26, 30 and 87; and c) singlevariable domains with 95% identity to said single variable domain withSEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides the polypeptide of paragraph [00623], whereinsaid polypeptide comprises four identical single variable domains andwherein said single variable domain is selected from the groupconsisting of a) singe variable domains with SEQ ID NOs: 1-5, 26, 30 and87; and b) single variable domains with at least 95% identity to atleast one single variable domain with SEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides the polypeptide of paragraph [00623] comprisingan amino sequence that is selected from the group consisting of a) anamino acid sequence of SEQ ID NO: 88; b) amino acid sequences with atleast 90% identity to said amino acid sequence with SEQ ID NO: 88; andc) amino acid sequences with 95% identity to said amino acid sequencewith SEQ ID NO: 88.

One embodiment provides the polypeptide of paragraph [00623] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequences with SEQ ID NO: 7, 10, 15, 18, 21, 28 and 32; b) aminoacid sequences with at least 90% identity to said amino acid sequencewith SEQ ID NO: 7, 10, 15, 18, 21, 28 and 32; and c) amino acidsequences with 95% identity to said amino acid sequence with SEQ ID NO:7, 10, 15, 18, 21, 28 and 32.

The polypeptide of paragraph [00623] comprising a humanized aminosequence that is selected from the group consisting of a) amino acidsequences with SEQ ID NO: 28; b) amino acid sequences with SEQ ID NO:32; and b) amino acid sequences with at least 95% identity to said aminoacid sequence with SEQ ID NO: 28 and 32.

One embodiment provides an isolated polypeptide comprising at least fivemonomers of a single variable domain that specifically binds to humanDR5.

One embodiment provides the polypeptide of paragraph [00630], whereinsaid polypeptide comprises five single variable domains and wherein saidsingle variable domain is selected from one, two, three, four or fivemonomers of the group consisting of a) singe variable domains with SEQID NOs: 1-5, 26, 30 and 87; b) single variable domains with at least 90%identity to said single variable domain with SEQ ID NOs: 1-5, 26, 30 and87; and c) single variable domains with at least 95% identity to saidsingle variable domain with SEQ ID NOs: 1-5, 26, 30 and 87 with at least90% identity.

One embodiment provides the polypeptide of paragraph [00630], whereinsaid polypeptide comprises five identical single variable domains andwherein said single variable domain is selected from the groupconsisting of a) singe variable domains with SEQ ID NOs: 1-5, 26, 30 and87; b) single variable domains with at least 90% identity to said singlevariable domain with SEQ ID NOs: 1-5, 26, 30 and 87; and b) singlevariable domains with 95% identity to said single variable domain withSEQ ID NOs: 1-5, 26, 30 and 87.

One embodiment provides the polypeptide of paragraph [00630], whereinsaid polypeptide comprises five single variable domains and wherein saidsingle variable domain is humanized, and said humanized domain isselected from the group consisting of a) singe variable domains with SEQID NOs: 26 and 30; b) single variable domains with at least 90% identityto at least one single variable domain with SEQ ID NOs: 26 and 30; andb) single variable domains with at least 95% identity to at least onesingle variable domain with SEQ ID NOs: 26 and 30.

One embodiment provides the polypeptide of paragraph [00630] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequences with SEQ ID NO: 8, 11, 16, 19, 22, 29 and 33; b) aminoacid sequences with at least 90% identity to said amino acid sequencewith SEQ ID NO: 8, 11, 16, 19, 22, 29 and 33; and c) amino acidsequences with 95% identity to said amino acid sequence with SEQ ID NO:8, 11, 16, 19, 22, 29 and 33.

One embodiment provides the polypeptide of paragraph [00630] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequence of SEQ ID NO: 29; b) amino acid sequences with at least90% identity to said amino acid sequence with SEQ ID NO: 29; and b)amino acid sequences with 95% identity to said amino acid sequence withSEQ ID NO: 29.

One embodiment provides the polypeptide of paragraph [00630] comprisingan amino sequence that is selected from the group consisting of a) aminoacid sequences with SEQ ID NO: 33; b) amino acid sequences with at least90% identity to said amino acid sequence with SEQ ID NO: 33; and b)amino acid sequences with at least 95% identity to said amino acidsequence with SEQ ID NO: 33.

One embodiment provides an isolated polypeptide comprising three or fouror five monomers of a single variable domain that specifically binds tohuman DR5 and wherein said polypeptide has an in vitro potency (IC₅₀)against a panel of at least 2 tumor cell lines selected from the groupconsisting of Colo205, Jurkat, Molt4, H2122, H226 and H2052 that isequal or lower than 100 nM.

One embodiment provides the polypeptide of paragraph [00637], whereinsaid polypeptide has an in vitro potency that is equal or lower than 10nM.

One embodiment provides the polypeptide of paragraph [00637], whereinsaid polypeptide has an in vitro potency that is equal or lower than 1nM.

One embodiment provides the polypeptide of paragraph [00637], whereinsaid polypeptide has an in vitro potency that is equal or lower than 100pM.

One embodiment provides the polypeptide of paragraphs [00637] to[00640], wherein said polypeptide has an in vitro potency (IC₅₀) againsta panel of at least 3 non-tumor cell lines selected from the groupconsisting of Malme-3, WI-38, ARPE-19, 184A1, Huvec, HAAE-1 that isequal of greater than 20 nM.

One embodiment provides the polypeptide of paragraphs [00637] to[00640], wherein said polypeptide has an in vitro potency (IC₅₀) againsta panel of at least 3 non-tumor cell lines selected from the groupconsisting of Malme-3, WI-38, ARPE-19, 184A1, Huvec, HAAE-1 that isequal of greater than 100 nM.

One embodiment provides the polypeptide of paragraphs [00637] to[00642], wherein said polypeptide comprises three or four or fivemonomers of a single variable domain and wherein said single variabledomain is selected from the group consisting of a) singe variabledomains with SEQ ID NOs: 1-5, 26, 30 and 87; and b) single variabledomains with at least 90% identity to said single variable domain withSEQ ID NOs: 1-5, 26, 30 and 87.

The polypeptide of paragraphs [00637] to [00642], wherein saidpolypeptide comprises three or four or five monomers of a singlevariable domain and wherein said single variable domain is humanized andselected from the group consisting of a) singe variable domains with SEQID NOs: 26 and 30; b) single variable domains with at least 90% identityto said single variable domain with SEQ ID NOs: 26 and 30; and b) singlevariable domains with at least 95% identity to said single variabledomain with SEQ ID NOs: 26 and 30.

One embodiment provides the polypeptide of paragraphs [00637] to[00642], wherein said polypeptide is selected from the group consistingof a) the polypeptide selected from any one or more of SEQ ID NOs: 27,28 and 29; b) a polypeptide with at least 95% identity to at least onepolypeptide selected from any one or more of SEQ ID NOs: 27, 28 and 29;c) the polypeptide selected from any one or more of SEQ ID NOs: 31, 32and 33; and d) a polypeptide with at least 95% identity to at least onepolypeptide selected from any one or more of SEQ ID NOs: 31, 32 and 33.

One embodiment provides an isolated polypeptide comprising an aminosequence that is selected from the group consisting of one or more aminoacid sequences of SEQ ID NO: 1 through 22, 26 through 33, 87 and 88.

One embodiment provides the isolated polypeptide according to any of thepreceding paragraphs [00612] to present, wherein said polypeptide doesnot fully or partially compete with the natural ligand of the DR5 in acompetitive binding assay.

One embodiment provides the isolated polypeptide according to any of thepreceding paragraphs [00612] to present, wherein said polypeptide doesfully or partially compete with the natural ligand of the DR5 in acompetitive binding assay.

One embodiment provides the isolated polypeptide according to paragraphs[00612], [00616], [00623], [00630], [00637] or [00648], comprising atleast one complementarity determining region (CDR) having at least 60,70, 80, 90, 95 or 100 percent sequence identity with at least one of theCDR regions depicted in any of SEQ ID NO: 41 to 44 (CDR1); SEQ ID NO: 51to 55 (CDR2); SEQ ID NO: 63 to 68 (CDR3), more preferably SEQ ID NO: 51to 55 and SEQ ID NO: 63 to 68.

One embodiment provides the isolated polypeptide according to paragraphs[00612], [00616], [00623], [00630], [00637] or [00648], comprising CDR1to CDR3 regions having at least 90, 95 or 100 percent sequence identityrespectively with the CDR regions depicted in any of SEQ ID NO: 41 to 44(CDR1); SEQ ID NO: 51 to 55 (CDR2); SEQ ID NO: 63 to 68 (CDR3).

One embodiment provides the isolated polypeptide according to any ofparagraphs [00612], [00616], [00623], [00630], [00637] or [00648],comprising or essentially consisting of an amino acid sequence having atleast 90, 95 or 100 percent sequence identity with any of sequences asdepicted in SEQ ID NO: 26 to 33.

One embodiment provides the isolated polypeptide according to any ofparagraphs [00612], [00616], [00623], [00630], [00637] or [00648],comprising a CDR3 region according to SEQ ID NO: 64 or SEQ ID NO: 66.

One embodiment provides the isolated polypeptide according to any ofparagraphs [00612], [00616], [00623], [00630], [00637] or [00648], aCDR1, CDR2 and CDR3 region identical respectively to the CDR regionsdepicted in SEQ ID NO: 41 to 44 (CDR1); SEQ ID NO: 51 to 55 (CDR2); SEQID NO: 63 to 68 (CDR3) or variant CDR regions with 1, 2, 3, 4 or 5 aminoacid substituted, deleted or inserted when compared to original CDRregions as depicted therein.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that is an immunoglobulin or afragment thereof.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that is a humanizedimmunoglobulin, a camelized immunoglobulin or an immunoglobulinobtainable by affinity optimization technique, or a fragment thereof.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of alight chain variable domain sequence, e.g., a V_(L)-sequence; or of aheavy chain variable domain sequence, e.g., a V_(H)-sequence.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of aheavy chain variable domain sequence that is derived from a conventionalfour-chain antibody or that essentially consists of a heavy chainvariable domain sequence that is derived from heavy chain antibody.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of adomain antibody, a single domain antibody, a dAb and a camelid antibodyor fragment, including but not limited to a V_(HH) sequence.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of aV_(HH) sequence.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of aV_(HH) sequence that, a) has at least 90% amino acid identity with atleast one of the amino acid sequences of SEQ ID NO's 1-22, 26-40, 87-88,and 102-103, in which for the purposes of determining the degree ofamino acid identity, the amino acid residues that form the CDR sequencesare disregarded; and in which:

b) optionally one or more of the amino acid positions 11, 37, 44, 45,47, 83, 84, 103, 104 and 108 according to Kabat numbering arehumaneered.

One embodiment provides the isolated polypeptide according to any one ormore of the preceding paragraphs [00612] to present that essentiallyconsists of a V_(HH) sequence that:

a) has at least 90% amino acid identity with at least one of the aminoacid sequences of SEQ ID NO's 1-22, 26-40, 87-88, and 102-103, in whichfor the purposes of determining the degree of amino acid identity, theamino acid residues that form the CDR sequences are disregarded; and inwhich

b) optionally the contacts between DR5 and the V_(HH) sequence areselected from:

-   -   i) the DR5 conformational epitope comprising the residues in        Table 29A;    -   ii) the DR5 conformational epitope comprising the residues in        Table 30A;    -   iii) the interaction surface of the V_(HH) amino acids as listed        in Table 29B; and    -   iv) the interaction surface of the V_(HH) amino acids as listed        in Table 30B.

One embodiment provides the isolated polypeptide according to anypreceding paragraphs [00612] to present that essentially consists of ahumanized V_(HH) sequence.

One embodiment provides the compound that comprises or essentiallyconsists of one or more polypeptide according to any of any precedingparagraphs [00612] to present, and optionally further comprises one ormore other groups, residues, moieties or binding units, wherein saidcompound is capable of enhancing cell apoptosis.

One embodiment provides the compound of paragraph [00663], which has anIC₅₀ less than 100 nM, preferably less than 10 nM, more preferably lessthan 1 nM, even more preferably less than 100 pM, e.g. below 10 pM as,e.g., measured in Colo205 cell survival assay.

One embodiment provides the compound of paragraphs [00663] or [00664],wherein the corresponding monovalent binding polypeptide is not aspotent as the multivalent polypeptide as measured, e.g., in Colo205 cellbased survival assay.

One embodiment provides the compound of any of paragraphs [00663] or[00665], wherein said at least three, four, five or more monovalentbinding polypeptides can each bind human DR5 in a monovalent format.

One embodiment provides the compound of any of paragraphs [00663] or[00665], wherein said at least three, four, five or more monovalentbinding polypeptides can each bind human DR5 in a monovalent format butdoes not bind to TRAIL-R3 and/or TRAIL-R4 receptors.

One embodiment provides the compound of any of paragraphs [00663] or[00665], wherein said at least three, four, five or more monovalentbinding polypeptide can each bind DR5 in a monovalent format andcompetes in a binding assay with natural TRAIL ligand.

One embodiment provides the compound of any of paragraphs [00663] or[00665], wherein said at least three, four, five or more monovalentbinding polypeptides are all directed against the same binding region ofhuman DR5.

One embodiment provides the compound of any of paragraphs [00663] or[00664], wherein said at least one monovalent binding polypeptide isdirected against one binding region of DR5 and at least one othermonovalent binding polypeptide is directed against another distinctbinding region of DR5 or a binding region of another DR5.

One embodiment provides the compound of any of paragraphs [00663] or[00664], wherein said at least three, four, five or more monovalentbinding polypeptides have substantially identical amino acid sequences.

One embodiment provides the compound of any of paragraphs [00663] or[00664], which essentially consists of a single chain polypeptide andwherein said at least three, four, five or more monovalent bindingsingle variable domains are linked together via peptidic linker.

One embodiment provides the compound of paragraph [00672], wherein saidlinker consists essentially of a peptide comprising between 5 and 50amino acid residues.

One embodiment provides the compound of any of paragraphs [00663] or[00665], wherein said one or more other groups, residues, moieties orbinding units are polypeptides, optionally linked via one or morelinkers.

One embodiment provides the compound of any one of paragraphs [00612] to[00674], wherein said one or more linkers are peptidic or polypeptidiclinkers.

One embodiment provides the compound of paragraph [00675], wherein saidone or more other groups, residues, moieties or binding units areselected from the group consisting of a domain antibody, a single domainantibody, a dAb and a camelid antibody or fragment thereof, includingbut not limited to a V_(HH) sequence.

One embodiment provides the compound of any of paragraphs [00663] to[00677], wherein said one or more other groups, residues, moieties orbinding units provide the compound with an increased half-life whenadministered in a mammalian organism, compared to the same compoundwithout said one or more other groups, residues, moieties or bindingunits.

One embodiment provides an isolated nucleic acid that encodes either (1)a polypeptide comprising the amino acid sequence of the polypeptide asdefined in any of paragraphs [00612]-[00662] or (2) a compound accordingto any of paragraphs [00663] to [00677].

One embodiment provides a host cell that expresses, or that undersuitable circumstances is capable of expressing the nucleic acid ofparagraph [00678].

One embodiment provides a method for producing the DR5 bindingpolypeptide of any of paragraphs [00612] to [00662], or the compound ofany of paragraphs [00663] to [00677], comprising:

a) expressing, in a suitable host cell or a non-human host organism, thenucleic acid of paragraph [00678]; and

b) isolating and/or purifying said DR5 binding polypeptide or compound.

One embodiment provides the polypeptide according to any of paragraphs[00612] to [00662] or compound according to any of paragraphs [00663] to[00677], for use as a drug.

One embodiment provides the polypeptide of paragraph [00681], for use asan anti-cancer therapeutic.

One embodiment provides a composition comprising at least a polypeptideof any of paragraphs [00612] to [00662], or a compound of any ofparagraphs [00663] to [00677] and at least one pharmaceuticallyacceptable carrier, diluent or excipient and/or adjuvant.

One embodiment provides a method for the prevention and/or treatment ofa disorder that can be treated by enhancement of cell apoptosis, saidmethod comprising administering, to a subject in need thereof, apharmaceutically effective amount of at least one polypeptide accordingto any of paragraphs [00612]-[00662], or of at least one compoundaccording to any of paragraphs [00663] to [00677].

One embodiment provides the method of paragraph [00684], wherein saiddisorder is a proliferative disease.

One embodiment provides the method of paragraph [00685], wherein saidproliferative disease is selected from:

a) one or more solid cancers selected from primary and metastaticcancers such as renal cell carcinoma, and cancers of the lung (e.g.,small cell lung cancer “SCLC” and non-small cell lung cancer “NSCLC”),pancreas, hematopoietic malignancy, glioma, astrocytoma, mesothelioma,colorectal cancers, prostate cancer, osteosarcoma, melanoma, lymphomalymphoma (including but not limited to Burkitt's Lymphoma), breastcancer, endometrial cancer, liver cancer, gastric cancer, skin cancer,ovarian cancer and squamous cell cancers of any origin (including butnot limited to squamous cell cancers of the lung, head and neck, breast,thyroid, cervix, skin, and/or esophageal); and

b) one or more liquid cancers selected from leukemias includingespecially a T-cell leukemia such as acute T-cell leukemia (T-ALL),acute B-cell leukemia (B-ALL), chronic myelogenous leukemia (CML), acutemyelogenous leukemia (AML), plasma cell myeloma and multiple myeloma(MM).

One embodiment provides the method of paragraph [00685], wherein saidproliferative disease is one or more non-cancer indications, saidindications comprising one or more of inflammatory and autoimmunediseases, such as systemic lupus erythematosus, Hashimoto's disease,rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome,pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome,Crohn's disease, autoimmune hemolytic anemia, sterility, myastheniagravis, multiple sclerosis, Basedow's disease, thromboticthrobocytopenia, thrombopenia purpurea, insulin-dependent diabetesmellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; globerula nephritis; and hypoplasticanemia.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is pancreatic cancer.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is T-ALL.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is mesothelioma.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is squamous cell carcinoma of any tissue origin.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is AML.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is melanoma.

One embodiment provides the method of paragraph [00686], wherein theproliferative disease is myeloma.

One embodiment provides a composition comprising the polypeptideaccording to any of paragraphs [00612]-[00662] or at least one compoundaccording to any of paragraphs [00663] to [00677], said composition incombination with at least one inhibitor of any one or more genesselected from cIAP1, cIAP2, XIAP, cFLIP and Bcl-xL.

One embodiment provides the composition of paragraph [00695], whereinthe inhibitor is an inhibitor of cIAP1.

One embodiment provides the composition of paragraph [00696] furthercomprising an inhibitor of MEK.

One embodiment provides the composition of paragraph [00696] furthercomprising an inhibitor of BRAF,

One embodiment provides the composition of paragraph [00695], whereinthe inhibitor is an inhibitor of XIAP.

One embodiment provides the composition of paragraph [00695], whereinthe inhibitor is an inhibitor of cFLIP.

One embodiment provides the composition of paragraph [00695], whereinthe inhibitor is a low molecular weight compound selected from LCL161and ABT263.

One embodiment provides the composition of paragraph [00695], whereinthe inhibitor is a shRNA.

One embodiment provides a method for using any one or more of thecompositions of paragraph [00683], the method comprising administeringto a subject in need thereof a pharmaceutically effected amount of saidcomposition or combination thereof for the treatment of a DR5 associateddisease.

One embodiment provides the method of paragraph [00703] wherein the DR5associated disease is selected from one or more of the following:

a) one or more proliferative diseases selected from solid and liquidcancers,

(i) wherein solid tumors include primary and metastatic cancers such asrenal cell carcinoma, and cancers of the lung (e.g., small cell lungcancer “SCLC” and non-small cell lung cancer “NSCLC”), pancreas,hematopoietic malignancy, glioma, astrocytoma, mesothelioma, colorectalcancers, prostate cancer, osteosarcoma, melanoma, lymphoma including butnot limited to Burkitt's lymphoma), breast cancer, endometrial cancer,liver cancer, gastric cancer, skin cancer, ovarian cancer and squamouscell cancers of any origin (including but not limited to squamous cellcancers of the lung, head and neck, breast, thyroid, cervix, skin,and/or esophageal); and

(ii) wherein liquid cancers comprise leukemias including especially aT-cell leukemia such as acute T-cell leukemia (T-ALL), acute B-cellleukemia (B-ALL), chronic myelogenous leukemia (CML), acute myelogenousleukemia (AML), plasma cell myeloma and multiple myeloma (MM); and

b) non-cancer indications comprising one or more of inflammatory andautoimmune diseases, such as systemic lupus erythematosus, Hashimoto'sdisease, rheumatoid arthritis, graft-versus-host disease, Sjogren'ssyndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture'ssyndrome, Crohn's disease, autoimmune hemolytic anemia; sterility,myasthenia gravis, multiple sclerosis, Basedow's disease, thromboticthrobocytopenia, thrombopenia purpurea, insulin-dependent diabetesmellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; globerula nephritis; and hypoplasticanemia.

One embodiment provides the isolated polypeptides according to any oneor more of the preceding paragraphs [00612] to present wherein the CDRsare calculated according to either Kabat and/or Chothia rules.

One embodiment provides the isolated polypeptides according to any oneor more of the preceding paragraphs [00612] through the present whereinthe polypeptide binds one or more epitopes as provided in Tables 29Aand/or 30A, and/or comprises the interactive surface residues asprovided in Tables 29B and/or 30B.

The following claims of the invention are non-limiting. Certainvariations to the invention may be contemplated that are within theabilities of one skilled in the art, including but not limited tochanges in formulations, delivery, humanization, expression systems andthe like. Such variations are considered to be within the scope of theinvention.

What is claimed is:
 1. A method of inducing apoptosis in a cell ortissue in a patient, comprising the method of administering to thepatient an isolated polypeptide comprising at least one monomer of asingle variable domain comprising the sequence of the complementaritydetermining region 3 (CDR3) sequence of SEQ ID NO: 66, the sequence ofthe complementarity determining region 2 (CDR2) sequence of SEQ ID NO:53, and the sequence of the complementarity determining region 1 (CDR1)sequence of SEQ ID NO: 43, Wherein the polypeptide is capable of bindingto DR5 on a surface of the cell or tissue and inducing apoptosis.
 2. Themethod of claim 1, wherein the cell or tissue is a cancer orproliferative disease.
 3. The method of claim 2, wherein the cell ortissue is selected from: a) one or more solid cancers selected fromprimary and metastatic cancers, renal cell carcinoma, and cancers of thelung, pancreas, hematopoietic malignancy, glioma, astrocytoma,mesothelioma, colorectal cancers, prostate cancer, osteosarcoma,melanoma, lymphoma lymphoma, breast cancer, endometrial cancer, livercancer, gastric cancer, skin cancer, ovarian cancer and squamous cellcancers of any origin, squamous cell cancers of the lung, head and neck,breast, thyroid, cervix, skin, and/or esophageal; and b) one or moreliquid cancers selected from leukemias, a T-cell leukemia such as acuteT-cell leukemia (T-ALL), acute B-cell leukemia (B-ALL), chronicmyelogenous leukemia (CML), acute myelogenous leukemia (AML), plasmacell myeloma and multiple myeloma (MM); and c) one or more non-cancerindications, said indications comprising one or more of inflammatory andautoimmune diseases, systemic lupus erythematosus, Hashimoto's disease,rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome,pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome,Crohn's disease, autoimmune hemolytic anemia, sterility, myastheniagravis, multiple sclerosis, Basedow's disease, thromboticthrobocytopenia, thrombopenia purpurea, insulin-dependent diabetesmellitus, allergy; asthma, atopic disease; arteriosclerosis;myocarditis; cardiomyopathy; globerula nephritis; and hypoplasticanemia.
 4. The method of claim 2, wherein the proliferative disease ispancreatic cancer.
 5. The method of claim 2, wherein the proliferativedisease is T-ALL.
 6. The method of claim 2, wherein the proliferativedisease is mesothelioma.
 7. The method of claim 2, wherein theproliferative disease is squamous cell carcinoma of any tissue origin.8. The method of claim 2, wherein the proliferative disease is AML. 9.The method of claim 2, wherein the proliferative disease is melanoma.10. The method of claim 2, wherein the proliferative disease is myeloma.11. The method of claim 1, wherein the single variable domain comprises:(a) the sequence of SEQ ID NO: 30; or (b) the sequence of SEQ ID NO: 30wherein one or more of the amino acid positions 1-30, 36-49, 66-97and/or 112-122 have been substituted, modified or deleted and/or whereinone or more amino acids have been inserted between any amino acids at1-30, 36-49, 66-97 and/or 112-122.
 12. The method of claim 1, whereinthe isolated polypeptide comprises at least three, at least four, or atleast five monomeric subunits of a single variable domain thatspecifically binds to human DR5, wherein said subunits are linked by alinker, and wherein the variable domain comprises the sequence of thecomplementarity determining region 3 (CDR3) sequence of SEQ ID NO: 66,the sequence of the complementarity determining region 2 (CDR2) sequenceof SEQ ID NO: 53, and the sequence of the complementarity determiningregion 1 (CDR1) sequence of SEQ ID NO:
 43. 13. The method of claim 12,wherein said single variable domain comprises the sequence of SEQ ID NO:30.
 14. The method of claim 13, wherein the single variable domaincomprises the amino acid sequence of SEQ ID NO: 30 wherein one or moreof the amino acid positions 1-30, 36-49, 66-97 and/or 112-122 have beensubstituted, modified or deleted, and/or wherein one or more amino acidshave been inserted between any amino acids at 1-30, 36-49, 66-97 and/or112-122.
 15. The method of claim 1, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO: 5, 20, 21, 22, 30, 31, 32, 33, or87.
 16. The method of claim 1, wherein the polypeptide has: (a) theamino acid sequence of SEQ ID NO: 30; or (b) the amino acid sequence ofSEQ ID NO: 30, in which one or more of the amino acid positions 11, 37,44, 45, 47, 83, 84, 112, 113 and 117 of SEQ ID NO: 30 are humaneered,substituted, modified or deleted and/or wherein one or more amino acidshave been inserted between any amino acids at 1-30, 36-49, 66-97 and/or112-122, wherein the isolated polypeptide specifically binds to humanDR5.
 17. The method of claim 1, wherein the polypeptide has: (a) thesequence of SEQ ID NO: 30; and/or (b) the sequence of SEQ ID NO: 30wherein one or more of the amino acid positions 1-30, 36-49, 66-97and/or 112-122 have been substituted, modified or deleted, and/orwherein one or more amino acids have been inserted between any aminoacids at 1-30, 36-49, 66-97 and/or 112-122, and wherein the isolatedpolypeptide is capable of enhancing cell apoptosis and wherein theisolated polypeptide specifically binds to human DR5.
 18. The methodaccording to claim 17, wherein the polypeptide comprises at least three,four, five or more copies of the sequence of SEQ ID NO: 30 wherein oneor more of the amino acid positions 1-30, 36-49, 66-97 and/or 112-122have been substituted, modified or deleted, and/or wherein one or moreamino acids have been inserted between any amino acids at 1-30, 36-49,66-97 and/or 112-122, and wherein the copies constitute monovalentbinding polypeptides that are all directed against the same bindingregion of human DR5.
 19. The method of claim 1, wherein the polypeptidecomprises at least three, four, five or more copies of the sequence ofSEQ ID NO: 30 wherein one or more of the amino acid positions 1-30,36-49, 66-97 and/or 112-122 have been substituted, modified or deletedand/or wherein one or more amino acids have been inserted between anyamino acids at 1-30, 36-49, 66-97 and/or 112-122, wherein the isolatedpolypeptide is capable of enhancing cell apoptosis and wherein thecopies constitute monovalent binding polypeptides that are all directedagainst the same binding region of human DR5, and wherein the saidisolated polypeptide specifically binds to human DR5.
 20. The method ofclaim 1, wherein the polypeptide comprises the CDR1 sequence of SEQ IDNO: 43, the CDR2 sequence of SEQ ID NO: 53, and the CDR3 sequence of SEQID NO: 66, wherein the polypeptide is capable of contacting, within 5Angstroms, amino acids H86, S88, E89, D90, D93, I95, K98, Q101, D102,L111, F112, R115, and R118 of a DR5 polypeptide of SEQ ID NO:
 89. 21.The method of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO: 30.